Dendritic enriched secreted lymphocyte activation molecule

ABSTRACT

The present invention relates to a novel human protein called Dendritic Enriched Secreted Lymphocyte Activation Molecule, and isolated polynucleotides encoding this protein. Also provided are vectors, host cells, antibodies, and recombinant methods for producing this human protein. The invention further relates to diagnostic and therapeutic methods useful for diagnosing and treating disorders related to this novel human protein.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.10/062,523, filed Feb. 5, 2002, which claims benefit under 35 U.S.C. §119(e) of U.S. Provisional Application Ser. No. 60/267,523, filed Feb.6, 2001 and which is a continuation-in-part of International PatentApplication Serial No. PCT/US00/21130, filed Aug. 3, 2000, which claimsbenefit under 35 U.S.C. § 119(e) of U.S. Provisional Application Ser.No. 60/190,062, filed Mar. 17, 2000 and which is a continuation-in-partof U.S. application Ser. No. 09/369,248, filed Aug. 5, 1999, now U.S.Pat. No. 6,620,912, which is a continuation-in-part of U.S. applicationSer. No. 09/244,110 filed Feb. 4, 1999, which claims benefit under 35U.S.C. § 119(e) of U.S. Provisional Application Ser. No. 60/073,962,filed Feb. 6, 1998, and 60/078,572, filed Mar. 19, 1998, each of whichis hereby incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present invention relates to a novel human gene encoding apolypeptide which is a member of the Secreted Lymphocyte ActivationMolecule (SLAM) family. More specifically, the present invention relatesto a polynucleotide encoding a novel human polypeptide named DendriticEnriched Secreted Lymphocyte Activation Molecule, or “D-SLAM.” Thisinvention also relates to D-SLAM polypeptides, as well as vectors, hostcells, antibodies directed to D-SLAM polypeptides, and the recombinantmethods for producing the same. Also provided are diagnostic methods fordetecting disorders related to the immune system, and therapeuticmethods for treating, diagnosing, detecting, and/or preventing suchdisorders. The invention further relates to screening methods foridentifying agonists and antagonists of D-SLAM activity.

BACKGROUND OF THE INVENTION

A member of the immunoglobulin gene superfamily, SLAM is rapidly inducedafter activation of naive T- and B-cells. (Cocks, B. G., “A NovelReceptor Involved in T-Cell Activation,” Nature 376:260-263 (1995);Aversa, G., “Engagement of the Signaling Lymphocytic Activation Molecule(SLAM) on Activated T Cells Results in Il-2-Independent, CyclosporinA-Sensitive T Cell Proliferation and IFN-gamma Production,” J. Immun.4036-4044 (1997).) A multifunctional 70 kDa glycoprotein, SLAM causesproliferation and differentiation of immune cells. (Punnonen, J.,“Soluble and Membrane-bound Forms of Signaling Lymphocytic ActivationMolecule (SLAM) Induce Proliferation and Ig Synthesis by Activated HumanB Lymphocytes,” J. Exp. Med. 185:993-1004 (1997).) To elicit an immuneresponse, both a secreted form of SLAM, as well as a membrane boundSLAM, are thought to interact.

It is also known that dendritic cells (DC) are the principal antigenpresenting cells involved in primary immune responses; their majorfunction is to obtain antigen in tissues, migrate to lymphoid organs,and activate T cells. (Mohamadzadeh, M. et al., J. Immunol. 156:3102-3106 (1996).) In fact, DC are usually the first immune cells toarrive at sites of inflammation on mucous membranes. (See, e.g.,Weissman, D. et al., J. Immunol. 155:4111-4117 (1995).) DC are alsoknown to directly interact with B cells. There is a constant need toidentify new polypeptide factors which may mediate interactions betweenDC and T cells and/or B cells, leading to the activation and/orproliferation of immune cells. To date, however, SLAM molecules have notbeen identified on DC cells.

Thus, there is a need for polypeptides that affect the proliferation,activation, survival, and/or differentiation of immune cells, such as T-and B-cells, since disturbances of such regulation may be involved indisorders relating to immune system. Therefore, there is a need foridentification and characterization of such human polypeptides which canplay a role in detecting, preventing, ameliorating or correcting suchdisorders.

SUMMARY OF THE INVENTION

The present invention relates to a novel polynucleotide and the encodedpolypeptide of D-SLAM. Moreover, the present invention relates tovectors, host cells, antibodies, and recombinant methods for producingthe polypeptides and polynucleotides. Also provided are diagnosticmethods for detecting and/or diagnosing disorders relates to thepolypeptides, and therapeutic methods for treating and/or preventingsuch disorders. The invention further relates to screening methods foridentifying binding partners of D-SLAM.

In accordance with one embodiment of the present invention, there isprovided a novel mature D-SLAM polypeptide, as well as biologicallyactive and diagnostically or therapeutically useful fragments, analogsand derivatives thereof.

In a preferred embodiment, the invention provides for D-SLAMpolypeptides and/or polynucleotides (including, but not limited to,fragments and/or variants thereof), and/or agonists thereof, which canbe used, for example, to inhibit B-cell proliferation.

In a further preferred embodiment, the invention provides for D-SLAMpolypeptides (including, but not limited to, soluble forms of D-SLAM)and/or D-SLAM antagonists, which can be used, for example, to enhanceB-cell proliferation.

In accordance with another embodiment of the present invention, thereare provided isolated nucleic acid molecules encoding human D-SLAM,including mRNAs, DNAs, cDNAs, genomic DNAs as well as analogs andbiologically active and diagnostically or therapeutically usefulfragments and derivatives thereof.

The present invention provides isolated nucleic acid moleculescomprising, or alternatively, consisting of, a polynucleotide encoding acytokine that are structurally similar to Secreted Lymphocyte ActivationMolecules (SLAM) and related cytokines and have similar biologicaleffects and activities. This cytokine is named D-SLAM and the inventionincludes D-SLAM polypeptides having at least a portion of the amino acidsequence in FIGS. 1A, 1B, 1C, and 1D (SEQ ID NO:2) or amino acidsequence encoded by the cDNA clone (HDPJO39) deposited on Feb. 6, 1998assigned ATCC number 209623. The nucleotide sequence determined bysequencing the deposited D-SLAM clone, which is shown in FIGS. 1A, 1B,1C, and 1D (SEQ ID NO:1), contains an open reading frame encoding acomplete polypeptide of 285 amino acid residues including an N-terminalmethionine (i.e., amino acid residues 1-285 of SEQ ID NO:2), a predictedsignal peptide of about 22 amino acid residues (i.e., amino acidresidues 1-22 of SEQ ID NO:2), a predicted mature form of about 263amino acids (i.e., amino acid residues 23-285 of SEQ ID NO:2), and adeduced molecular weight for the complete protein of about 34.2 kDa.

Thus, one embodiment of the invention provides an isolated nucleic acidmolecule comprising, or alternatively consisting of, a polynucleotidehaving a nucleotide sequence selected from the group consisting of: (a)a nucleotide sequence encoding a full-length D-SLAM polypeptide havingthe complete amino acid sequence in FIGS. 1A, 1B, 1C, and 1D (SEQ IDNO:2) or as encoded by the cDNA clone contained in the deposit havingATCC accession number 209623; (b) a nucleotide sequence encoding thepredicted extracellular domain of the D-SLAM polypeptide having theamino acid sequence at positions 23 to 232 in FIGS. 1A, 1B, 1C, and 1D(SEQ ID NO:2) or as encoded by the clone contained in the deposit havingATCC accession number 209623; (c) a nucleotide sequence encoding afragment of the polypeptide of (b) having D-SLAM functional activity(e.g., biological activity); (d) a nucleotide sequence encoding apolypeptide comprising the D-SLAM intracellular domain (predicted toconstitute amino acid residues from about 256 to about 285 in FIGS. 1A,1B, 1C, and 1D (SEQ ID NO:2)) or as encoded by the clone contained inthe deposit having ATCC accession number 209623; (e) a nucleotidesequence encoding a polypeptide comprising the D-SLAM transmembranedomain (predicted to constitute amino acid residues from about 233 toabout 255 in FIGS. 1A, 1B, 1C, and 1D (SEQ ID NO:2) or as encoded by thecDNA clone contained in the deposit having ATCC accession number 209623;(f) a nucleotide sequence encoding a soluble D-SLAM polypeptide havingthe extracellular and intracellular domains but lacking thetransmembrane domain; and (g) a nucleotide sequence complementary to anyof the nucleotide sequences in (a), (b), (c), (d), (e) or (f) above.

Further embodiments of the invention include isolated nucleic acidmolecules that comprise, or alternatively consist of, a polynucleotidehaving a nucleotide sequence at least 80%, 85% or 90% identical, andmore preferably at least 95%, 96%, 97%, 98% or 99% identical, to any ofthe nucleotide sequences in (a), (b), (c), (d), (e), (f) or (g) above,or a polynucleotide which hybridizes under stringent hybridizationconditions to a polynucleotide in (a), (b), (c), (d), (e), (f) or (g)above. This polynucleotide which hybridizes does not hybridize understringent hybridization conditions to a polynucleotide having anucleotide sequence consisting of only A residues or of only T residues.

In additional embodiments, the nucleic acid molecules of the inventioncomprise, or alternatively consist of, a polynucleotide which encodesthe amino acid sequence of an epitope-bearing portion of a D-SLAMpolypeptide having an amino acid sequence in (a), (b), (c), (d), (e),(f) or (g) above. A further nucleic acid embodiment of the inventionrelates to an isolated nucleic acid molecule comprising, oralternatively consisting of, a polynucleotide which encodes the aminoacid sequence of a D-SLAM polypeptide having an amino acid sequencewhich contains at least one amino acid addition, substitution, and/ordeletion but not more than 50 amino acid additions, substitutions and/ordeletions, even more preferably, not more than 40 amino acid additions,substitutions, and/or deletions, still more preferably, not more than 30amino acid additions, substitutions, and/or deletions, and still evenmore preferably, not more than 20 amino acid additions, substitutions,and/or deletions. Of course, in order of ever-increasing preference, itis highly preferable for a polynucleotide which encodes the amino acidsequence of a D-SLAM polypeptide to have an amino acid sequence whichcontains not more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 or 1-100, 1-50,1-25, 1-20, 1-15, 1-10, or 1-5 amino acid additions, substitutionsand/or deletions. Conservative substitutions are preferable.

The present invention also relates to recombinant vectors, which includethe isolated nucleic acid molecules of the present invention, and tohost cells containing the recombinant vectors, as well as to methods ofmaking such vectors and host cells and for using them for production ofD-SLAM polypeptides by recombinant techniques.

In accordance with a further embodiment of the present invention, thereis provided a process for producing such polypeptides by recombinanttechniques comprising culturing recombinant prokaryotic and/oreukaryotic host cells, containing a D-SLAM nucleic acid sequence of theinvention, under conditions promoting expression of said polypeptide andsubsequent recovery of said polypeptide.

The invention further provides an isolated D-SLAM polypeptidecomprising, or alternatively consisting of, an amino acid sequenceselected from the group consisting of: (a) the amino acid sequence ofthe full-length D-SLAM polypeptide having the complete amino acidsequence shown in FIGS. 1A, 1B, 1C, and 1D (i.e., positions 1-285 of SEQID NO:2) or as encoded by the cDNA plasmid contained in the deposithaving ATCC accession number 209623; (b) the amino acid sequence of thefull-length D-SLAM polypeptide having the complete amino acid sequenceshown in SEQ ID NO:2 excepting the N-terminal methionine (i.e.,positions 2 to 285 of SEQ ID NO:2); (c) a fragment of the polypeptide of(b) having D-SLAM functional activity (e.g., biological activity (suchas, for example, inhibition of B-cell proliferation)); (d) the aminoacid sequence of the predicted extracellular domain of the D-SLAMpolypeptide having the amino acid sequence at positions 23 to 232 inFIGS. 1A, 1B, 1C, and 1D (SEQ ID NO:2) or as encoded by the cDNA plasmidcontained in the deposit having ATCC accession number 209623; (e) theamino acid sequence of the D-SLAM intracellular domain (predicted toconstitute amino acid residues from about 256 to about 285 in FIGS. 1A,1B, 1C, and 1D (SEQ ID NO:2)) or as encoded by the cDNA plasmidcontained in the deposit having ATCC accession number 209623; (f) theamino acid sequence of the D-SLAM transmembrane domain (predicted toconstitute amino acid residues from about 233 to about 255 in FIGS. 1A,1B, 1C, and 1D (SEQ ID NO:2)) or as encoded by the cDNA plasmidcontained in the deposit having ATCC accession number 209623; (g) theamino acid sequence of the soluble D-SLAM polypeptide having theextracellular and intracellular domains but lacking the transmembranedomain, wherein each of these domains is defined above; and (h)fragments of the polypeptide of (a), (b), (c), (d), (e), (f) or (g). Thepolypeptides of the present invention also include polypeptides havingan amino acid sequence at least 80% identical, more preferably at least85% or 90% identical, and still more preferably 95%, 96%, 97%, 98% or99% identical to those described in (a), (b), (c), (d), (e), (f) or (g)above, as well as polypeptides having an amino acid sequence with atleast 80%, 85%, or 90% similarity, and more preferably at least 95%similarity, to those above. Additional embodiments of the inventionrelates to polypeptides which comprise, or alternatively consist of, theamino acid sequence of an epitope-bearing portion of a D-SLAMpolypeptide having an amino acid sequence described in (a), (b), (c),(d), (e), (f), (g) or (h) above. Polypeptides having the amino acidsequence of an epitope-bearing portion of a D-SLAM polypeptide of theinvention include portions of such polypeptides with at least 4, atleast 5, at least 6, at least 7, at least 8, and preferably at least 9,at least 10, at least 11, at least 12, at least 13, at least 14, atleast 15, at least 20, at least 25, at least 30, at least 40, at least50, and more preferably at least about 30 amino acids to about 50 aminoacids, although epitope-bearing polypeptides of any length up to andincluding the entire amino acid sequence of a polypeptide of theinvention described above also are included in the invention.

The present invention also encompasses the above polynucleotidesequences fused to a heterologous polynucleotide sequence. Polypeptidesencoded by these polynucleotides and nucleic acid molecules are alsoencompassed by the invention.

Certain non-exclusive embodiments of the invention relate to apolypeptide which has the amino acid sequence of an epitope-bearingportion of a D-SLAM polypeptide having an amino acid sequence describedin (a), (b), (c), (d), (e), (f), (g), (h) or (i) above. In otherembodiments, the invention provides an isolated antibody that bindsspecifically (i.e., uniquely) to a D-SLAM polypeptide having an aminoacid sequence described in (a), (b), (c), (d), (e), (f), (g), (h) or (i)above.

The invention further provides methods for isolating antibodies thatbind specifically (i.e., uniquely) to a D-SLAM polypeptide having anamino acid sequence as described herein. Such antibodies are usefuldiagnostically or therapeutically as described below.

The invention also provides for pharmaceutical compositions comprisingsoluble D-SLAM polypeptides, particularly human D-SLAM polypeptides,and/or anti-D-SLAM antibodies which may be employed, for instance, totreat, prevent, prognose and/or diagnose tumor and tumor metastasis,infections by bacteria, viruses and other parasites, immunodeficiencies,inflammatory diseases, lymphadenopathy, autoimmune diseases, graftversus host disease, stimulate peripheral tolerance, destroy sometransformed cell lines, mediate cell activation, survival andproliferation, to mediate immune regulation and inflammatory responses,and to enhance or inhibit immune responses.

In certain embodiments, soluble D-SLAM polypeptides of the invention,and/or antagonists of D-SLAM, are administered, to treat, prevent,prognose and/or diagnose an immunodeficiency (e.g., severe combinedimmunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminasedeficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton'sdisease, congenital agammaglobulinemia, X-linked infantileagammaglobulinemia, acquired agammaglobulinemia, adult onsetagammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia,hypogammaglobulinemia, transient hypogammaglobulinemia of infancy,unspecified hypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymphoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency) or conditions associated with animmunodeficiency.

In a specific embodiment, D-SLAM polypeptides or polynucleotides of theinvention, or antagonists thereof, is administered to treat, prevent,prognose and/or diagnose common variable immunodeficiency.

In a specific embodiment, D-SLAM polypeptides or polynucleotides of theinvention, or antagonists thereof, is administered to treat, prevent,prognose and/or diagnose X-linked agammaglobulinemia.

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists thereof, is administered to treat,prevent, prognose and/or diagnose severe combined immunodeficiency(SCID).

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists thereof, is administered to treat,prevent, prognose and/or diagnose Wiskott-Aldrich syndrome.

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists thereof, is administered to treat,prevent, prognose and/or diagnose X-linked Ig deficiency with hyper IgM.

In another embodiment, D-SLAM agonists are administered to treat,prevent, prognose and/or diagnose an autoimmune disease (e.g.,rheumatoid arthritis, systemic lupus erhythematosus, idiopathicthrombocytopenia purpura, autoimmune hemolytic anemia, autoimmuneneonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia,antiphospholipid syndrome, dermatitis, allergic encephalomyelitis,myocarditis, relapsing polychondritis, rheumatic heart disease,glomerulonephritis (e.g, IgA nephropathy), Multiple Sclerosis, Neuritis,Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g.,Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome,Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulindependent diabetes mellitis, and autoimmune inflammatory eye, autoimmunethyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis,Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, forexample, (a) Graves' Disease, (b) Myasthenia Gravis, and (c) insulinresistance, autoimmune hemolytic anemia, autoimmune thrombocytopenicpurpura, schleroderma with anti-collagen antibodies, mixed connectivetissue disease, polymyositis/dermatomyositis, pernicious anemia,idiopathic Addison's disease, infertility, glomerulonephritis such asprimary glomerulonephritis and IgA nephropathy, bullous pemphigoid,Sjogren's syndrome, diabetes millitus, and adrenergic drug resistance(including adrenergic drug resistance with asthma or cystic fibrosis),chronic active hepatitis, primary biliary cirrhosis, other endocrinegland failure, vitiligo, vasculitis, post-MI, cardiotomy syndrome,urticaria, atopic dermatitis, asthma, inflammatory myopathies, and otherinflammatory, granulamatous, degenerative, and atrophic disorders) orconditions associated with an autoimmune disease. In a specificpreferred embodiment, rheumatoid arthritis is treated, prevented,prognosed and/or diagnosed using D-SLAM and/or other agonists of theinvention. In another specific preferred embodiment, systemic lupuserythemosus is treated, prevented, prognosed, and/or diagnosed usingD-SLAM and/or other agonists of the invention. In another specificpreferred embodiment, idiopathic thrombocytopenia purpura is treated,prevented, prognosed, and/or diagnosed using D-SLAM and/or otheragonists of the invention. In another specific preferred embodiment IgAnephropathy is treated, prevented, prognosed and/or diagnosed usingD-SLAM and/or other agonists of the invention. In a preferredembodiment, the autoimmune diseases and disorders and/or conditionsassociated with the diseases and disorders recited above are treated,prevented, prognosed and/or diagnosed using D-SLAM.

The invention further provides compositions comprising a D-SLAMpolynucleotide, a D-SLAM polypeptide, and/or an anti-D-SLAM antibody,for administration to cells in vitro, to cells ex vivo, and to cells invivo, or to a multicellular organism. In preferred embodiments, thecompositions of the invention comprise a D-SLAM polynucleotide forexpression of a D-SLAM polypeptide in a host organism for treatment ofdisease. In a most preferred embodiment, the compositions of theinvention comprise a D-SLAM polynucleotide for expression of a D-SLAMpolypeptide in a host organism for treatment of an immunodeficiencyand/or conditions associated with an immunodeficiency. Particularlypreferred in this regard is expression in a human patient for treatmentof a dysfunction associated with aberrant endogenous activity of aD-SLAM gene.

The present invention also provides a screening method for identifyingcompounds capable of enhancing or inhibiting a cellular response inducedby D-SLAM which involves contacting cells which express D-SLAM with thecandidate compound, assaying a cellular response, and comparing thecellular response to a standard cellular response, the standard beingassayed when contact is made in absence of the candidate compound;whereby, an increased cellular response over the standard indicates thatthe compound is an agonist and a decreased cellular response over thestandard indicates that the compound is an antagonist.

In another embodiment, a method for identifying D-SLAM receptors isprovided, as well as a screening assay for agonists and antagonistsusing such receptors. This assay involves determining the effect acandidate compound has on D-SLAM binding to the D-SLAM receptor. Inparticular, the method involves contacting a D-SLAM receptor with aD-SLAM polypeptide of the invention and a candidate compound anddetermining whether D-SLAM polypeptide binding to the D-SLAM receptor isincreased or decreased due to the presence of the candidate compound.The agonists may be employed to prevent septic shock, inflammation,cerebral malaria, activation of the HIV virus, graft-host rejection,bone resorption, rheumatoid arthritis, cachexia (wasting ormalnutrition), immune system function, lymphoma, and autoimmunedisorders (e.g., rheumatoid arthritis and systemic lupus erythematosus).

The present inventors have discovered that D-SLAM is highly expressed indendritic cells and lymph node, and to a lesser extent in spleen,thymus, small intestine, PBLs, bone marrow, T cell lymphoma, and evenlesser extent in placenta and lung. For a number of disorders of thesetissues and cells, such as tumor and tumor metastasis, infection ofbacteria, viruses and other parasites, immunodeficiencies (e.g., chronicvariable immunodeficiency), septic shock, inflammation, cerebralmalaria, activation of the HIV virus, graft-host rejection, boneresorption, rheumatoid arthritis, autoimmune diseases (e.g., rheumatoidarthritis and systemic lupus erythematosus) and cachexia (wasting ormalnutrition) it is believed that D-SLAM polynucleotides and/orpolypeptides of the invention are useful for diagnosis, prevention,treatment, and/or amelioration. It is believed that significantly higheror lower levels of D-SLAM gene expression can be detected in certaintissues (e.g., bone marrow) or bodily fluids (e.g., serum, plasma,urine, synovial fluid or spinal fluid) taken from an individual havingsuch a disorder, relative to a “standard” D-SLAM gene expression level,i.e., the D-SLAM expression level in tissue or bodily fluids from anindividual not having the disorder. Thus, the invention provides adiagnostic method useful during diagnosis of a disorder, which involves:(a) assaying D-SLAM gene expression level in cells or body fluid of anindividual; (b) comparing the D-SLAM gene expression level with astandard D-SLAM gene expression level, whereby an increase or decreasein the assayed D-SLAM gene expression level compared to the standardexpression level is indicative of a disorder.

An additional embodiment of the invention is related to a method fortreating an individual in need of an increased or constitutive level ofD-SLAM activity in the body comprising administering to such anindividual a composition comprising a therapeutically effective amountof an isolated D-SLAM polypeptide of the invention or an agonistthereof.

Additionally, anti-D-SLAM antibodies (including anti-D-SLAM antibodyfragments) against the polypeptides of the invention may be used toquantitate or qualitate concentrations of cells of dendritic celllineage (e.g., dendritic cell related leukemias or lymphomas) expressingD-SLAM on their cell surfaces.

A still further embodiment of the invention is related to a method fortreating an individual in need of a decreased level of D-SLAM activityin the body comprising, administering to such an individual acomposition comprising a therapeutically effective amount of an D-SLAMantagonist. Preferred antagonists for use in the present invention areD-SLAM-specific antibodies.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D show the nucleotide sequence (SEQ ID NO:1) and the deducedamino acid sequence (SEQ ID NO:2) of D-SLAM. The predicted leadersequence located at about amino acids 1-22 is underlined.

FIG. 2 shows the regions of identity between the amino acid sequence ofthe D-SLAM protein and the translation product of the human SLAM(Accession No. gi/984969) (SEQ ID NO:3), determined by BLAST analysis.Identical amino acids between the two polypeptides are shaded, whileconservative amino acids are boxed. By examining the regions of aminoacids shaded and/or boxed, the skilled artisan can readily identifyconserved domains between the two polypeptides. These conserved domainsare preferred embodiments of the present invention.

FIG. 3 shows an analysis of the D-SLAM amino acid sequence. Alpha, beta,turn and coil regions; hydrophilicity and hydrophobicity; amphipathicregions; flexible regions; antigenic index and surface probability areshown, and all were generated using the default settings. In the“Antigenic Index or Jameson-Wolf” graph, the positive peaks indicatelocations of the highly antigenic regions of the D-SLAM protein, i.e.,regions from which epitope-bearing peptides of the invention can beobtained. The domains defined by these graphs are contemplated by thepresent invention. Tabular representation of the data summarizedgraphically in FIG. 3 can be found in Table 1.

The columns are labeled with the headings “Res”, “Position”, and RomanNumerals I-XIV. The column headings refer to the following features ofthe amino acid sequence presented in FIG. 3, and Table I: “Res”: aminoacid residue of SEQ ID NO:2 and FIGS. 1A and 1B; “Position”: position ofthe corresponding residue within SEQ ID NO:2 and FIGS. 1A and 1B; I:Alpha, Regions—Garnier-Robson; II: Alpha, Regions—Chou-Fasman; III:Beta, Regions—Garnier-Robson; IV: Beta, Regions—Chou-Fasman; V: Turn,Regions—Garnier-Robson; VI: Turn, Regions—Chou-Fasman; VII: Coil,Regions—Garnier-Robson; VIII: Hydrophilicity Plot—Kyte-Doolittle; IX:Hydrophobicity Plot—Hopp-Woods; X: Alpha, Amphipathic Regions—Eisenberg;XI: Beta, Amphipathic Regions—Eisenberg; XII: FlexibleRegions—Karplus-Schulz; XIII: Antigenic Index—Jameson-Wolf; and XIV:Surface Probability Plot—Emini.

DETAILED DESCRIPTION

Definitions

The following definitions are provided to facilitate understanding ofcertain terms used throughout this specification.

In the present invention, “isolated” refers to material removed from itsoriginal environment (e.g., the natural environment if it is naturallyoccurring), and thus is altered “by the hand of man” from its naturalstate. For example, an isolated polynucleotide could be part of a vectoror a composition of matter, or could be contained within a cell, andstill be “isolated” because that vector, composition of matter, orparticular cell is not the original environment of the polynucleotide.Further examples of isolated DNA molecules include recombinant DNAmolecules maintained in heterologous host cells or purified (partiallyor substantially) DNA molecules in solution. Isolated RNA moleculesinclude in vivo or in vitro RNA transcripts of the DNA molecules of thepresent invention. However, a nucleic acid contained in a clone that isa member of a library (e.g., a genomic or cDNA library) that has notbeen isolated from other members of the library (e.g., in the form of ahomogeneous solution containing the clone and other members of thelibrary) or a chromosome removed from a cell or a cell lysate (e.g., a“chromosome spread”, as in a karyotype), or a preparation of randomlysheared genomic DNA or a preparation of genomic DNA cut with one or morerestriction enzymes is not “isolated” for the purposes of thisinvention. As discussed further herein, isolated nucleic acid moleculesaccording to the present invention may be produced naturally,recombinantly, or synthetically.

In the present invention, a “secreted” D-SLAM protein refers to aprotein capable of being directed to the ER, secretory vesicles, or theextracellular space as a result of a signal sequence, as well as aD-SLAM protein released into the extracellular space without necessarilycontaining a signal sequence. If the D-SLAM secreted protein is releasedinto the extracellular space, the D-SLAM secreted protein can undergoextracellular processing to produce a “mature” D-SLAM protein. Releaseinto the extracellular space can occur by many mechanisms, includingexocytosis and proteolytic cleavage.

As used herein, a D-SLAM “polynucleotide” refers to a molecule having anucleic acid sequence contained in SEQ ID NO:1 or the cDNA containedwithin the clone deposited with the ATCC. For example, the D-SLAMpolynucleotide can contain the nucleotide sequence of the full lengthcDNA sequence, including the 5′ and 3′ untranslated sequences, thecoding region, with or without the signal sequence, the secreted proteincoding region, as well as fragments, epitopes, domains, and variants ofthe nucleic acid sequence. Moreover, as used herein, a D-SLAM“polypeptide” refers to a molecule having the translated amino acidsequence generated from the polynucleotide as broadly defined.

In specific embodiments, the polynucleotides of the invention are lessthan 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb in length.In a further embodiment, polynucleotides of the invention comprise atleast 15 contiguous nucleotides of D-SLAM coding sequence, but do notcomprise all or a portion of any D-SLAM intron. In another embodiment,the nucleic acid comprising D-SLAM coding sequence does not containcoding sequences of a genomic flanking gene (i.e., 5′ or 3′ to theD-SLAM gene in the genome).

In the present invention, the full length D-SLAM sequence identified asSEQ ID NO:1 was generated by overlapping sequences of the depositedclone (contig analysis). A representative clone containing all or mostof the sequence for SEQ ID NO:1 was deposited with the American TypeCulture Collection (“ATCC”) on Feb. 6, 1998, and was given the ATCCDeposit Number 209623. The ATCC is located at 10801 UniversityBoulevard, Manassas, Va. 20110-2209, USA. The ATCC deposit was madepursuant to the terms of the Budapest Treaty on the internationalrecognition of the deposit of microorganisms for purposes of patentprocedure.

A D-SLAM “polynucleotide” also includes those polynucleotides capable ofhybridizing, under stringent hybridization conditions, to sequencescontained in SEQ ID NO:1, the complement thereof, or the cDNA within thedeposited clone. “Stringent hybridization conditions” refers to anovernight incubation at 42° C. in a solution comprising 50% formamide,5×SSC (750 mM NaCl, 75 mM sodium citrate), 50 mM sodium phosphate (pH7.6), 5× Denhardt's solution, 10% dextran sulfate, and 20 μg/mldenatured, sheared salmon sperm DNA, followed by washing the filters in0.1×SSC at about 65° C.

Also contemplated are nucleic acid molecules that hybridize to theD-SLAM polynucleotides at moderately high stringency hybridizationconditions. Changes in the stringency of hybridization and signaldetection are primarily accomplished through the manipulation offormamide concentration (lower percentages of formamide result inlowered stringency); salt conditions, or temperature. For example,moderately high stringency conditions include an overnight incubation at37° C. in a solution comprising 6×SSPE (20×SSPE=3M NaCl; 0.2M NaH₂PO₄;0.02M EDTA, pH 7.4), 0.5% SDS, 30% formamide, 100 ug/ml salmon spermblocking DNA; followed by washes at 50° C. with 1×SSPE, 0.1% SDS. Inaddition, to achieve even lower stringency, washes performed followingstringent hybridization can be done at higher salt concentrations (e.g.5×SSC).

Note that variations in the above conditions may be accomplished throughthe inclusion and/or substitution of alternate blocking reagents used tosuppress background in hybridization experiments. Typical blockingreagents include Denhardt's reagent, BLOTTO, heparin, denatured salmonsperm DNA, and commercially available proprietary formulations. Theinclusion of specific blocking reagents may require modification of thehybridization conditions described above, due to problems withcompatibility.

Of course, a polynucleotide which hybridizes only to polyA+ sequences(such as any 3′ terminal polyA+ tract of a cDNA shown in the sequencelisting), or to a complementary stretch of T (or U) residues, would notbe included in the definition of “polynucleotide,” since such apolynucleotide would hybridize to any nucleic acid molecule containing apoly (A) stretch or the complement thereof (e.g., practically anydouble-stranded cDNA clone).

The D-SLAM polynucleotide can be composed of any polyribonucleotide orpolydeoxribonucleotide, which may be unmodified RNA or DNA or modifiedRNA or DNA. For example, D-SLAM polynucleotides can be composed ofsingle- and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions, single- and double-stranded RNA, and RNA thatis mixture of single- and double-stranded regions, hybrid moleculescomprising DNA and RNA that may be single-stranded or, more typically,double-stranded or a mixture of single- and double-stranded regions. Inaddition, the D-SLAM polynucleotides can be composed of triple-strandedregions comprising RNA or DNA or both RNA and DNA. D-SLAMpolynucleotides may also contain one or more modified bases or DNA orRNA backbones modified for stability or for other reasons. “Modified”bases include, for example, tritylated bases and unusual bases such asinosine. A variety of modifications can be made to DNA and RNA; thus,“polynucleotide” embraces chemically, enzymatically, or metabolicallymodified forms.

D-SLAM polypeptides can be composed of amino acids joined to each otherby peptide bonds or modified peptide bonds, i.e., peptide isosteres, andmay contain amino acids other than the 20 gene-encoded amino acids. TheD-SLAM polypeptides may be modified by either natural processes, such asposttranslational processing, or by chemical modification techniqueswhich are well known in the art. Such modifications are well describedin basic texts and in more detailed monographs, as well as in avoluminous research literature. Modifications can occur anywhere in theD-SLAM polypeptide, including the peptide backbone, the amino acidside-chains and the amino or carboxyl termini. It will be appreciatedthat the same type of modification may be present in the same or varyingdegrees at several sites in a given D-SLAM polypeptide. Also, a givenD-SLAM polypeptide may contain many types of modifications. D-SLAMpolypeptides may be branched, for example, as a result ofubiquitination, and they may be cyclic, with or without branching.Cyclic, branched, and branched cyclic D-SLAM polypeptides may resultfrom posttranslation natural processes or may be made by syntheticmethods. Modifications include, but are not limited to, acetylation,acylation, ADP-ribosylation, amidation, biotinylation, covalentattachment of flavin, covalent attachment of a heme moiety, covalentattachment of a nucleotide or nucleotide derivative, covalent attachmentof a lipid or lipid derivative, covalent attachment ofphosphotidylinositol, cross-linking, cyclization, derivatization byknown protecting/blocking groups, disulfide bond formation,demethylation, formation of covalent cross-links, formation of cysteine,formation of pyroglutamate, formylation, gamma-carboxylation,glycosylation, GPI anchor formation, hydroxylation, iodination, linkageto an antibody molecule or other cellular ligand, methylation,myristoylation, oxidation, pegylation, proteolytic processing (e.g.,cleavage), phosphorylation, prenylation, racemization, selenoylation,sulfation, transfer-RNA mediated addition of amino acids to proteinssuch as arginylation, and ubiquitination. (See, for instance,PROTEINS—STRUCTURE AND MOLECULAR PROPERTIES, 2nd Ed., T. E. Creighton,W. H. Freeman and Company, New York (1993); POSTTRANSLATIONAL COVALENTMODIFICATION OF PROTEINS, B. C. Johnson, Ed., Academic Press, New York,pgs. 1-12 (1983); Seifter et al., Meth Enzymol 182:626-646 (1990);Rattan et al., Ann NY Acad Sci 663:48-62 (1992).) Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited, to specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin, etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of prokaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein. In addition,polypeptides of the invention may be modified by iodination.

In one embodiment, D-SLAM polypeptides of the invention may also belabeled with biotin. In other related embodiments, biotinylated D-SLAMpolypeptides of the invention may be used, for example, as an imagingagent or as a means of identifying one or more D-SLAM receptor(s) orother coreceptor or coligand molecules.

Also provided by the invention are chemically modified derivatives ofD-SLAM which may provide additional advantages such as increasedsolubility, stability and in vivo or in vitro circulating time of thepolypeptide, or decreased immunogenicity (see U.S. Pat. No. 4,179,337).The chemical moieties for derivitization may be selected from watersoluble polymers such as polyethylene glycol, ethylene glycol/propyleneglycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcoholand the like. The polypeptides may be modified at random positionswithin the molecule, or at predetermined positions within the moleculeand may include one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, thedegree or lack of antigenicity and other known effects of thepolyethylene glycol to a therapeutic protein or analog). For example,the polyethylene glycol may have an average molecular weight of about200, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500,6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000,11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500,16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000,25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,600,75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.

As noted above, the polyethylene glycol may have a branched structure.Branched polyethylene glycols are described, for example, in U.S. Pat.No. 5,643,575; Morpurgo et al., Appl. Biochem. Biotechnol. 56:59-72(1996); Vorobjev et al., Nucleosides Nucleotides 18:2745-2750 (1999);and Caliceti et al., Bioconjug. Chem. 10:638-646 (1999), the disclosuresof each of which are incorporated herein by reference.

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include, for example, lysineresidues and the N-terminal amino acid residues; those having a freecarboxyl group may include aspartic acid residues, glutamic acidresidues, and the C-terminal amino acid residue. Sulfhydryl groups mayalso be used as a reactive group for attaching the polyethylene glycolmolecules. Preferred for therapeutic purposes is attachment at an aminogroup, such as attachment at the N-terminus or lysine group.

As suggested above, polyethylene glycol may be attached to proteins vialinkage to any of a number of amino acid residues. For example,polyethylene glycol can be linked to a proteins via covalent bonds tolysine, histidine, aspartic acid, glutamic acid, or cysteine residues.One or more reaction chemistries may be employed to attach polyethyleneglycol to specific amino acid residues (e.g., lysine, histidine,aspartic acid, glutamic acid, or cysteine) of the protein or to morethan one type of amino acid residue (e.g., lysine, histidine, asparticacid, glutamic acid, cysteine and combinations thereof) of the protein.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration, one may selectfrom a variety of polyethylene glycol molecules (by molecular weight,branching, etc.), the proportion of polyethylene glycol molecules toprotein (or peptide) molecules in the reaction mix, the type ofpegylation reaction to be performed, and the method of obtaining theselected N-terminally pegylated protein. The method of obtaining theN-terminally pegylated preparation (i.e., separating this moiety fromother monopegylated moieties if necessary) may be by purification of theN-terminally pegylated material from a population of pegylated proteinmolecules. Selective proteins chemically modified at the N-terminusmodification may be accomplished by reductive alkylation which exploitsdifferential reactivity of different types of primary amino groups(lysine versus the N-terminal) available for derivatization in aparticular protein. Under the appropriate reaction conditions,substantially selective derivatization of the protein at the N-terminuswith a carbonyl group containing polymer is achieved.

As indicated above, pegylation of the proteins of the invention may beaccomplished by any number of means. For example, polyethylene glycolmay be attached to the protein either directly or by an interveninglinker. Linkerless systems for attaching polyethylene glycol to proteinsare described in Delgado et al., Crit. Rev. Thera. Drug Carrier Sys.9:249-304 (1992); Francis et al., Intern. J of Hematol. 68:1-18 (1998);U.S. Pat. No. 4,002,531; U.S. Pat. No. 5,349,052; WO 95/06058; and WO98/32466, the disclosures of each of which are incorporated herein byreference.

One system for attaching polyethylene glycol directly to amino acidresidues of proteins without an intervening linker employs tresylatedMPEG, which is produced by the modification of monmethoxy polyethyleneglycol (MPEG) using tresylchloride (ClSO₂CH₂CF₃). Upon reaction ofprotein with tresylated MPEG, polyethylene glycol is directly attachedto amine groups of the protein. Thus, the invention includesprotein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a2,2,2-trifluoreothane sulphonyl group.

Polyethylene glycol can also be attached to proteins using a number ofdifferent intervening linkers. For example, U.S. Pat. No. 5,612,460, theentire disclosure of which is incorporated herein by reference,discloses urethane linkers for connecting polyethylene glycol toproteins. Protein-polyethylene glycol conjugates wherein thepolyethylene glycol is attached to the protein by a linker can also beproduced by reaction of proteins with compounds such asMPEG-succinimidylsuccinate, MPEG activated with1,1′-carbonyldiimidazole, MPEG-2,4,5-trichloropenylcarbonate,MPEG-p-nitrophenolcarbonate, and various MPEG-succinate derivatives. Anumber additional polyethylene glycol derivatives and reactionchemistries for attaching polyethylene glycol to proteins are describedin WO 98/32466, the entire disclosure of which is incorporated herein byreference. Pegylated protein products produced using the reactionchemistries set out herein are included within the scope of theinvention.

The number of polyethylene glycol moieties attached to each protein ofthe invention (i.e., the degree of substitution) may also vary. Forexample, the pegylated proteins of the invention may be linked, onaverage, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or morepolyethylene glycol molecules. Similarly, the average degree ofsubstitution within ranges such as 1-3,2-4, 3-5,4-6, 5-7,6-8, 7-9,8-10,9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18-20polyethylene glycol moieties per protein molecule. Methods fordetermining the degree of substitution are discussed, for example, inDelgado et al., Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992).

The D-SLAM polypeptides can be recovered and purified by known methodswhich include, but are not limited to, ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification.

“SEQ ID NO:1” refers to a D-SLAM polynucleotide sequence while “SEQ IDNO:2” refers to a D-SLAM polypeptide sequence.

A D-SLAM polypeptide “having biological activity” refers to polypeptidesexhibiting activity similar, but not necessarily identical to, anactivity of a D-SLAM polypeptide, including mature forms, (e.g., bindmonocyte derived dendritic cells, upregulate cell surface markersindicative of dendritic cell maturation or activation (such as, forexample, HLA-DR, CD54, CD86, CD83), inhibit B-cell proliferation,inhibit BLyS induced cell proliferative activity, bind an antibody whichbinds D-SLAM) as measured in a particular biological assay, with orwithout dose dependency. In the case where dose dependency does exist,it need not be identical to that of the D-SLAM polypeptide, but rathersubstantially similar to the dose-dependence in a given activity ascompared to the D-SLAM polypeptide (i.e., the candidate polypeptide willexhibit greater activity or not more than about 25-fold less and,preferably, not more than about tenfold less activity, and mostpreferably, not more than about three-fold less activity relative to theD-SLAM polypeptide.)

D-SLAM Polynucleotides and Polypeptides

Clone HDPJO39 was isolated from a dendritic cell cDNA library. Thisclone contains the entire coding region identified as SEQ ID NO:2. Thedeposited clone contains a cDNA having a total of 3220 nucleotides,which encodes a predicted open reading frame of 285 amino acid residues.(See FIGS. 1A-1D.) The open reading frame begins at a N-terminalmethionine located at nucleotide position 92, and ends at a stop codonat nucleotide position 947. The predicted molecular weight of the D-SLAMprotein should be about 34.2 kDa.

Subsequent Northern analysis also showed D-SLAM expression in dendriticcells, T cell lymphoma, lymph node, spleen, thymus, small intestine, anduterus tissues, a pattern consistent with hematopoietic specificexpression. Expression is highest in tissues involved in immunerecognition, consistent with the enriched expression in dendritic cellsand APC's. A single primary transcript of approximately 3.5-4.0 kb isobserved, with a minor transcript of 7-9 kb that likely represents anunprocessed RNA precursor. The expression of the major 3.5-4 kbtranscript is highest in lymph node, spleen, thymus, and, to a lesserdegree, in small intestine. The highest expression of the 7-9 kbtranscript is observed in the uterus.

Using BLAST analysis, SEQ ID NO:2 was found to be homologous to membersof the Secreted Lymphocyte Activation Molecule (SLAM) family.Particularly, SEQ ID NO:2 contains domains homologous to the translationproduct of the human mRNA for SLAM (Accession No. gi/984969) (FIG. 2)(SEQ ID NO:3), including the following conserved domains: (a) apredicted transmembrane domain located at about amino acids 233-255; (b)a predicted extracellular domain located at about amino acids 23-232;and (c) a predicted intracellular domain located at about amino acids256-285. These polypeptide fragments of D-SLAM are specificallycontemplated in the present invention. Because SLAM (Accession No.gi/984969) is thought to be important in the activation andproliferation of T- and B-cells, the homology between SLAM (AccessionNo. gi/984969) and D-SLAM suggests that D-SLAM may also be involved inthe activation and proliferation of T- and B-cells.

Moreover, the encoded polypeptide has a predicted leader sequencelocated at about amino acids 1-22. (See FIGS. 1A-1D.) Also shown inFIGS. 1A-1D, the predicted secreted form of D-SLAM encompasses aboutamino acids 23-232. These polypeptide fragments of D-SLAM arespecifically contemplated in the present invention.

The D-SLAM nucleotide sequence identified as SEQ ID NO:1 was assembledfrom partially homologous (“overlapping”) sequences obtained from thedeposited clone, and in some cases, from additional related DNA clones.The overlapping sequences were assembled into a single contiguoussequence of high redundancy (usually three to five overlapping sequencesat each nucleotide position), resulting in a final sequence identifiedas SEQ ID NO:1.

Therefore, SEQ ID NO:1 and the translated SEQ ID NO:2 are sufficientlyaccurate and otherwise suitable for a variety of uses well known in theart and described further below. For instance, SEQ ID NO:1 is useful fordesigning nucleic acid hybridization probes that will detect nucleicacid sequences contained in SEQ ID NO:1 or the cDNA contained in thedeposited clone. These probes will also hybridize to nucleic acidmolecules in biological samples, thereby enabling a variety of forensicand diagnostic methods of the invention. Similarly, polypeptidesidentified from SEQ ID NO:2 may be used to generate antibodies whichbind specifically to D-SLAM.

Nevertheless, DNA sequences generated by sequencing reactions cancontain sequencing errors. The errors exist as misidentifiednucleotides, or as insertions or deletions of nucleotides in thegenerated DNA sequence. The erroneously inserted or deleted nucleotidescause frame shifts in the reading frames of the predicted amino acidsequence. In these cases, the predicted amino acid sequence divergesfrom the actual amino acid sequence, even though the generated DNAsequence may be greater than 99.9% identical to the actual DNA sequence(for example, one base insertion or deletion in an open reading frame ofover 1000 bases).

Accordingly, for those applications requiring precision in thenucleotide sequence or the amino acid sequence, the present inventionprovides not only the generated nucleotide sequence identified as SEQ IDNO:1 and the predicted translated amino acid sequence identified as SEQID NO:2, but also a sample of plasmid DNA containing a human cDNA ofD-SLAM deposited with the ATCC. The nucleotide sequence of the depositedD-SLAM clone can readily be determined by sequencing the deposited clonein accordance with known methods. The predicted D-SLAM amino acidsequence can then be verified from such deposits. Moreover, the aminoacid sequence of the protein encoded by the deposited clone can also bedirectly determined by peptide sequencing or by expressing the proteinin a suitable host cell containing the deposited human D-SLAM cDNA,collecting the protein, and determining its sequence.

The present invention also relates to the D-SLAM gene corresponding toSEQ ID NO:1, SEQ ID NO:2, or the deposited clone. The D-SLAM gene can beisolated in accordance with known methods using the sequence informationdisclosed herein. Such methods include preparing probes or primers fromthe disclosed sequence and identifying or amplifying the D-SLAM genefrom appropriate sources of genomic material.

Also provided in the present invention are species homologs of D-SLAM.Species homologs may be isolated and identified by making suitableprobes or primers from the sequences provided herein and screening asuitable nucleic acid source for the desired homologue.

The D-SLAM polypeptides can be prepared in any suitable manner. Suchpolypeptides include isolated naturally occurring polypeptides,recombinantly produced polypeptides, synthetically producedpolypeptides, or polypeptides produced by a combination of thesemethods. Means for preparing such polypeptides are well understood inthe art.

The D-SLAM polypeptides may be in the form of the secreted protein,including the mature form, or may be a part of a larger protein, such asa fusion protein (see below). It is often advantageous to include anadditional amino acid sequence which contains secretory or leadersequences, pro-sequences, sequences which aid in purification, such asmultiple histidine residues, or an additional sequence for stabilityduring recombinant production.

D-SLAM polypeptides are preferably provided in an isolated form, andpreferably are substantially purified. A recombinantly produced versionof a D-SLAM polypeptide, including the secreted polypeptide, can besubstantially purified by the one-step method described in Smith andJohnson, Gene 67:31-40 (1988). D-SLAM polypeptides also can be purifiedfrom natural or recombinant sources using antibodies of the inventionraised against the D-SLAM protein in methods which are well known in theart.

Polynucleotide and Polypeptide Variants

“Variant” refers to a polynucleotide or polypeptide differing from theD-SLAM polynucleotide or polypeptide, but retaining essential propertiesthereof. Generally, variants are overall closely similar, and, in manyregions, identical to the D-SLAM polynucleotide or polypeptide.

By a polynucleotide having a nucleotide sequence at least, for example,95% “identical” to a reference nucleotide sequence of the presentinvention, it is intended that the nucleotide sequence of thepolynucleotide is identical to the reference sequence except that thepolynucleotide sequence may include up to five point mutations per each100 nucleotides of the reference nucleotide sequence encoding the D-SLAMpolypeptide. In other words, to obtain a polynucleotide having anucleotide sequence at least 95% identical to a reference nucleotidesequence, up to 5% of the nucleotides in the reference sequence may bedeleted or substituted with another nucleotide, or a number ofnucleotides up to 5% of the total nucleotides in the reference sequencemay be inserted into the reference sequence. The query sequence may bean entire sequence shown of SEQ ID NO:1, the ORF (open reading frame),or any fragment specified as described herein.

As a practical matter, whether any particular nucleic acid molecule orpolypeptide is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to a nucleotide sequence of the presence invention can bedetermined conventionally using known computer programs. A preferredmethod for determining the best overall match between a query sequence(a sequence of the present invention) and a subject sequence, alsoreferred to as a global sequence alignment, can be determined using theFASTDB computer program based on the algorithm of Brutlag et al. (Comp.App. Biosci. (1990) 6:237-245.) In a sequence alignment the query andsubject sequences are both DNA sequences. An RNA sequence can becompared by converting U's to T's. The result of said global sequencealignment is in percent identity. Preferred parameters used in a FASTDBalignment of DNA sequences to calculate percent identity are:Matrix=Unitary, k-tuple=4, Mismatch Penalty=1, Joining Penalty=30,Randomization Group Length=0, Cutoff Score=1, Gap Penalty=5, Gap SizePenalty 0.05, Window Size=500 or the length of the subject nucleotidesequence, whichever is shorter.

If the subject sequence is shorter than the query sequence because of 5′or 3′ deletions, not because of internal deletions, a manual correctionmust be made to the results. This is because the FASTDB program does notaccount for 5′ and 3′ truncations of the subject sequence whencalculating percent identity. For subject sequences truncated at the 5′or 3′ ends, relative to the query sequence, the percent identity iscorrected by calculating the number of bases of the query sequence thatare 5′ and 3′ of the subject sequence, which are not matched/aligned, asa percent of the total bases of the query sequence. Whether a nucleotideis matched/aligned is determined by results of the FASTDB sequencealignment. This percentage is then subtracted from the percent identity,calculated by the above FASTDB program using the specified parameters,to arrive at a final percent identity score. This corrected score iswhat is used for the purposes of the present invention. Only basesoutside the 5′ and 3′ bases of the subject sequence, as displayed by theFASTDB alignment, which are not matched/aligned with the query sequence,are calculated for the purposes of manually adjusting the percentidentity score.

For example, a 90 base subject sequence is aligned to a 100 base querysequence to determine percent identity. The deletions occur at the 5′end of the subject sequence and therefore, the FASTDB alignment does notshow a matched/alignment of the first 10 bases at 5′ end. The 10unpaired bases represent 10% of the sequence (number of bases at the 5′and 3′ ends not matched/total number of bases in the query sequence) so10% is subtracted from the percent identity score calculated by theFASTDB program. If the remaining 90 bases were perfectly matched thefinal percent identity would be 90%. In another example, a 90 basesubject sequence is compared with a 100 base query sequence. This timethe deletions are internal deletions so that there are no bases on the5′ or 3′ of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only bases 5′ and 3′ of the subjectsequence which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

By a polypeptide having an amino acid sequence at least, for example,95% “identical” to a query amino acid sequence of the present invention,it is intended that the amino acid sequence of the subject polypeptideis identical to the query sequence except that the subject polypeptidesequence may include up to five amino acid alterations per each 100amino acids of the query amino acid sequence. In other words, to obtaina polypeptide having an amino acid sequence at least 95% identical to aquery amino acid sequence, up to 5% of the amino acid residues in thesubject sequence may be inserted, deleted, (indels) or substituted withanother amino acid. These alterations of the reference sequence mayoccur at the amino or carboxy terminal positions of the reference aminoacid sequence or anywhere between those terminal positions, interspersedeither individually among residues in the reference sequence or in oneor more contiguous groups within the reference sequence.

As a practical matter, whether any particular polypeptide is at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, forinstance, the amino acid sequences shown in SEQ ID NO:2 or to the aminoacid sequence encoded by deposited DNA clone can be determinedconventionally using known computer programs. A preferred method fordetermining the best overall match between a query sequence (a sequenceof the present invention) and a subject sequence, also referred to as aglobal sequence alignment, can be determined using the FASTDB computerprogram based on the algorithm of Brutlag et al. (Comp. App. Biosci.(1990) 6:237-245). In a sequence alignment the query and subjectsequences are either both nucleotide sequences or both amino acidsequences. The result of said global sequence alignment is in percentidentity. Preferred parameters used in a FASTDB amino acid alignmentare: Matrix=PAM 0, k-tuple=2, Mismatch Penalty=1, Joining Penalty=20,Randomization Group Length=0, Cutoff Score=1, Window Size=sequencelength, Gap Penalty=5, Gap Size Penalty=0.05, Window Size=500 or thelength of the subject amino acid sequence, whichever is shorter.

If the subject sequence is shorter than the query sequence due to N- orC-terminal deletions, not because of internal deletions, a manualcorrection must be made to the results. This is because the FASTDBprogram does not account for N- and C-terminal truncations of thesubject sequence when calculating global percent identity. For subjectsequences truncated at the N- and C-termini, relative to the querysequence, the percent identity is corrected by calculating the number ofresidues of the query sequence that are N- and C-terminal of the subjectsequence, which are not matched/aligned with a corresponding subjectresidue, as a percent of the total bases of the query sequence. Whethera residue is matched/aligned is determined by results of the FASTDBsequence alignment. This percentage is then subtracted from the percentidentity, calculated by the above FASTDB program using the specifiedparameters, to arrive at a final percent identity score. This finalpercent identity score is what is used for the purposes of the presentinvention. Only residues to the N- and C-termini of the subjectsequence, which are not matched/aligned with the query sequence, areconsidered for the purposes of manually adjusting the percent identityscore. That is, only query residue positions outside the farthest N- andC-terminal residues of the subject sequence.

For example, a 90 amino acid residue subject sequence is aligned with a100 residue query sequence to determine percent identity. The deletionoccurs at the N-terminus of the subject sequence and therefore, theFASTDB alignment does not show a matching/alignment of the first 10residues at the N-terminus. The 10 unpaired residues represent 10% ofthe sequence (number of residues at the N- and C-termini notmatched/total number of residues in the query sequence) so 10% issubtracted from the percent identity score calculated by the FASTDBprogram. If the remaining 90 residues were perfectly matched the finalpercent identity would be 90%. In another example, a 90 residue subjectsequence is compared with a 100 residue query sequence. This time thedeletions are internal deletions so there are no residues at the N- orC-termini of the subject sequence which are not matched/aligned with thequery. In this case the percent identity calculated by FASTDB is notmanually corrected. Once again, only residue positions outside the N-and C-terminal ends of the subject sequence, as displayed in the FASTDBalignment, which are not matched/aligned with the query sequence aremanually corrected for. No other manual corrections are to made for thepurposes of the present invention.

The D-SLAM variants may contain alterations in the coding regions,non-coding regions, or both. Especially preferred are polynucleotidevariants containing alterations which produce silent substitutions,additions, or deletions, but do not alter the properties or activitiesof the encoded polypeptide. Nucleotide variants produced by silentsubstitutions due to the degeneracy of the genetic code are preferred.Moreover, variants in which 5-10, 1-5, or 1-2 amino acids aresubstituted, deleted, or added in any combination are also preferred.D-SLAM polynucleotide variants can be produced for a variety of reasons,e.g., to optimize codon expression for a particular host (change codonsin the human mRNA to those preferred by a bacterial host such as E.coli).

Naturally occurring D-SLAM variants are called “allelic variants,” andrefer to one of several alternate forms of a gene occupying a givenlocus on a chromosome of an organism. (Genes II, Lewin, B., ed., JohnWiley & Sons, New York (1985).) These allelic variants can vary ateither the polynucleotide and/or polypeptide level. Alternatively,non-naturally occurring variants may be produced by mutagenesistechniques or by direct synthesis.

Using known methods of protein engineering and recombinant DNAtechnology, variants may be generated to improve or alter thecharacteristics of the D-SLAM polypeptides. For instance, one or moreamino acids can be deleted from the N-terminus or C-terminus of thesecreted protein without substantial loss of biological function. Theauthors of Ron et al., J. Biol. Chem. 268: 2984-2988 (1993), reportedvariant KGF proteins having heparin binding activity even after deleting3, 8, or 27 amino-terminal amino acid residues. Similarly, Interferongamma exhibited up to ten times higher activity after deleting 8-10amino acid residues from the carboxy terminus of this protein. (Dobeliet al., J. Biotechnology 7:199-216 (1988).)

Moreover, ample evidence demonstrates that variants often retain abiological activity similar to that of the naturally occurring protein.For example, Gayle and coworkers (J. Biol. Chem. 268:22105-22111 (1993))conducted extensive mutational analysis of human cytokine IL-1a. Theyused random mutagenesis to generate over 3,500 individual IL-1a mutantsthat averaged 2.5 amino acid changes per variant over the entire lengthof the molecule. Multiple mutations were examined at every possibleamino acid position. The investigators found that “[m]ost of themolecule could be altered with little effect on either [binding orbiological activity].” (See, Abstract.) In fact, only 23 unique aminoacid sequences, out of more than 3,500 nucleotide sequences examined,produced a protein that significantly differed in activity fromwild-type.

Furthermore, even if deleting one or more amino acids from theN-terminus or C-terminus of a polypeptide results in modification orloss of one or more biological functions, other biological activitiesmay still be retained. For example, the ability of a deletion variant toinduce and/or to bind antibodies which recognize the secreted form willlikely be retained when less than the majority of the residues of thesecreted form are removed from the N-terminus or C-terminus. Whether aparticular polypeptide lacking N- or C-terminal residues of a proteinretains such immunogenic activities can readily be determined by routinemethods described herein and otherwise known in the art.

Thus, the invention further includes D-SLAM polypeptide variants whichshow substantial biological activity. Such variants include deletions,insertions, inversions, repeats, and substitutions selected according togeneral rules known in the art so as have little effect on activity.

The present application is directed to nucleic acid molecules at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleicacid sequences disclosed herein, (e.g., encoding a polypeptide havingthe amino acid sequence of an N and/or C terminal deletion disclosedbelow as m-n of SEQ ID NO:2), irrespective of whether they encode apolypeptide having D-SLAM functional activity. This is because evenwhere a particular nucleic acid molecule does not encode a polypeptidehaving D-SLAM functional activity, one of skill in the art would stillknow how to use the nucleic acid molecule, for instance, as ahybridization probe or a polymerase chain reaction (PCR) primer. Uses ofthe nucleic acid molecules of the present invention that do not encode apolypeptide having D-SLAM functional activity include, inter alia, (1)isolating a D-SLAM gene or allelic or splice variants thereof in a cDNAlibrary; (2) in situ hybridization (e.g., “FISH”) to metaphasechromosomal spreads to provide precise chromosomal location of theD-SLAM gene, as described in Verma et al., Human Chromosomes: A Manualof Basic Techniques, Pergamon Press, New York (1988); and (3) NorthernBlot analysis for detecting D-SLAM mRNA expression in specific tissues.

Preferred, however, are nucleic acid molecules having sequences at least80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleicacid sequences disclosed herein, which do, in fact, encode a polypeptidehaving D-SLAM functional activity. By “a polypeptide having D-SLAMfunctional activity” is intended polypeptides capable of exhibitingactivity similar, but not necessarily identical, to a known functionalactivity of the D-SLAM polypeptides of the present invention (e.g.,complete (full-length) D-SLAM, mature D-SLAM and soluble D-SLAM (e.g.,having sequences contained in the extracellular domain of D-SLAM) asmeasured, for example, in a particular immunoassay or biological assay.Such functional activities include, but are not limited to, biologicalactivity (e.g., ability to bind monocyte derived dendritic cells,ability to upregulate cell surface markers indicative of dendritic cellmaturation or activation (such as, for example, HLA-DR, CD54, CD86,CD83), ability to inhibit B-cell proliferation, ability to inhibit BLySinduced cell proliferative activity, antigenicity (ability to bind orcompete with a D-SLAM polypeptide for binding to an anti-D-SLAMantibody), immunogenicity (ability to generate an antibody whichspecifically binds to a D-SLAM polypeptide), ability to form multimerswith D-SLAM polypeptides of the invention, ability to formheteromultimers, ability to bind to a receptor or ligand). For example,a D-SLAM functional activity can routinely be measured by determiningthe ability of a D-SLAM polypeptide to bind a D-SLAM ligand. D-SLAMfunctional activity may also be measured by determining the ability of apolypeptide, such as cognate ligand which is free or expressed on a cellsurface, to induce cells expressing the polypeptide.

Of course, due to the degeneracy of the genetic code, one of ordinaryskill in the art will immediately recognize that a large number of thenucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of thedeposited cDNA, the nucleic acid sequence shown in FIG. 1 (SEQ ID NO:1),or fragments thereof, will encode polypeptides “having D-SLAM functionalactivity.” In fact, since degenerate variants of any of these nucleotidesequences all encode the same polypeptide, in many instances, this willbe clear to the skilled artisan even without performing the abovedescribed comparison assay. It will be further recognized in the artthat, for such nucleic acid molecules that are not degenerate variants,a reasonable number will also encode a polypeptide having D-SLAMfunctional activity. This is because the skilled artisan is fully awareof amino acid substitutions that are either less likely or not likely tosignificantly effect protein function (e.g., replacing one aliphaticamino acid with a second aliphatic amino acid), as further describedbelow.

For example, guidance concerning how to make phenotypically silent aminoacid substitutions is provided in Bowie, J. U. et al., Science247:1306-1310 (1990), wherein the authors indicate that there are twomain strategies for studying the tolerance of an amino acid sequence tochange.

The first strategy exploits the tolerance of amino acid substitutions bynatural selection during the process of evolution. By comparing aminoacid sequences in different species, conserved amino acids can beidentified. These conserved amino acids are likely important for proteinfunction. In contrast, the amino acid positions where substitutions havebeen tolerated by natural selection indicates that these positions arenot critical for protein function. Thus, positions tolerating amino acidsubstitution could be modified while still maintaining biologicalactivity of the protein.

The second strategy uses genetic engineering to introduce amino acidchanges at specific positions of a cloned gene to identify regionscritical for protein function. For example, site directed mutagenesis oralanine-scanning mutagenesis (introduction of single alanine mutationsat every residue in the molecule) can be used. (Cunningham and Wells,Science 244:1081-1085 (1989).) The resulting mutant molecules can thenbe tested for biological activity.

As the authors state, these two strategies have revealed that proteinsare surprisingly tolerant of amino acid substitutions. The authorsfurther indicate which amino acid changes are likely to be permissive atcertain amino acid positions in the protein. For example, most buried(within the tertiary structure of the protein) amino acid residuesrequire nonpolar side chains, whereas few features of surface sidechains are generally conserved. Moreover, tolerated conservative aminoacid substitutions involve replacement of the aliphatic or hydrophobicamino acids Ala, Val, Leu and Ile; replacement of the hydroxyl residuesSer and Thr; replacement of the acidic residues Asp and Glu; replacementof the amide residues Asn and Gln, replacement of the basic residuesLys, Arg, and His; replacement of the aromatic residues Phe, Tyr, andTrp, and replacement of the small-sized amino acids Ala, Ser, Thr, Met,and Gly.

For example, site directed changes at the amino acid level of D-SLAM canbe made by replacing a particular amino acid with a conservative aminoacid. Preferred conservative mutations include: M1 replaced with A, G,I, L, S, T, or V; V2 replaced with A, G, I, L, S, T, or M; M3 replacedwith A, G, I, L, S, T, or V; R4 replaced with H, or K; L6 replaced withA, G, I, S, T, M, or V; W7 replaced with F, or Y; S8 replaced with A, G,I, L, T, M, or V; L9 replaced with A, G, I, S, T, M, or V; L10 replacedwith A, G, I, S, T, M, or V; L11 replaced with A, G, I, S, T, M, or V;W12 replaced with F, or Y; E13 replaced with D; A14 replaced with G, I,L, S, T, M, or V; L15 replaced with A, G, I, S, T, M, or V; L16 replacedwith A, G, I, S, T, M, or V; I18 replaced with A, G, L, S, T, M, or V;T19 replaced with A, G, I, L, S, M, or V; V20 replaced with A, G, I, L,S, T, or M; T21 replaced with A, G, I, L, S, M, or V; G22 replaced withA, I, L, S, T, M, or V; A23 replaced with G, I, L, S, T, M, or V; Q24replaced with N; V25 replaced with A, G, I, L, S, T, or M; L26 replacedwith A, G, I, S, T, M, or V; S27 replaced with A, G, I, L, T, M, or V;K28 replaced with H, or R; V29 replaced with A, G, I, L, S, T, or M; G30replaced with A, I, L, S, T, M, or V; G31 replaced with A, I, L, S, T,M, or V; S32 replaced with A, G, I, L, T, M, or V; V33 replaced with A,G, I, L, S, T, or M; L34 replaced with A, G, I, S, T, M, or V; L35replaced with A, G, I, S, T, M, or V; V36 replaced with A, G, I, L, S,T, or M; A37 replaced with G, I, L, S, T, M, or V; A38 replaced with G,I, L, S, T, M, or V; R39 replaced with H, or K; G42 replaced with A, I,L, S, T, M, or V; F43 replaced with W, or Y; Q44 replaced with N; V45replaced with A, G, I, L, S, T, or M; R46 replaced with H, or K; E47replaced with D; A48 replaced with G, I, L, S, T, M, or V; I49 replacedwith A, G, L, S, T, M, or V; W50 replaced with F, or Y; R51 replacedwith H, or K; S52 replaced with A, G, I, L, T, M, or V; L53 replacedwith A, G, I, S, T, M, or V; W54 replaced with F, or Y; S56 replacedwith A, G, I, L, T, M, or V; E57 replaced with D; E58 replaced with D;L59 replaced with A, G, I, S, T, M, or V; L60 replaced with A, G, I, S,T, M, or V; A61 replaced with G, I, L, S, T, M, or V; T62 replaced withA, G, I, L, S, M, or V; F63 replaced with W, or Y; F64 replaced with W,or Y; R65 replaced with H, or K; G66 replaced with A, I, L, S, T, M, orV; S67 replaced with A, G, I, L, T, M, or V; L68 replaced with A, G, I,S, T, M, or V; E69 replaced with D; T70 replaced with A, G, I, L, S, M,or V; L71 replaced with A, G, I, S, T, M, or V; Y72 replaced with F, orW; H73 replaced with K, or R; S74 replaced with A, G, I, L, T, M, or V;R75 replaced with H, or K; F76 replaced with W, or Y; L77 replaced withA, G, I, S, T, M, or V; G78 replaced with A, I, L, S, T, M, or V; R79replaced with H, or K; A80 replaced with G, I, L, S, T, M, or V; Q81replaced with N; L82 replaced with A, G, I, S, T, M, or V; H83 replacedwith K, or R; S84 replaced with A, G, I, L, T, M, or V; N85 replacedwith Q; L86 replaced with A, G, I, S, T, M, or V; S87 replaced with A,G, I, L, T, M, or V; L88 replaced with A, G, I, S, T, M, or V; E89replaced with D; L90 replaced with A, G, I, S, T, M, or V; G91 replacedwith A, I, L, S, T, M, or V; L93 replaced with A, G, I, S, T, M, or V;E94 replaced with D; S95 replaced with A, G, I, L, T, M, or V; G96replaced with A, I, L, S, T, M, or V; D97 replaced with E; S98 replacedwith A, G, I, L, T, M, or V; G99 replaced with A, I, L, S, T, M, or V;N100 replaced with Q; F101 replaced with W, or Y; S102 replaced with A,G, I, L, T, M, or V; V103 replaced with A, G, I, L, S, T, or M; L104replaced with A, G, I, S, T, M, or V; M105 replaced with A, G, I, L, S,T, or V; V106 replaced with A, G, I, L, S, T, or M; D107 replaced withE; T108 replaced with A, G, I, L, S, M, or V; R109 replaced with H, orK; G110 replaced with A, I, L, S, T, M, or V; Q111 replaced with N; W113replaced with F, or Y; T114 replaced with A, G, I, L, S, M, or V; Q115replaced with N; T116 replaced with A, G, I, L, S, M, or V; L117replaced with A, G, I, S, T, M, or V; Q118 replaced with N; L119replaced with A, G, I, S, T, M, or V; K120 replaced with H, or R; V121replaced with A, G, I, L, S, T, or M; Y122 replaced with F, or W; D123replaced with E; A124 replaced with G, I, L, S, T, M, or V; V125replaced with A, G, I, L, S, T, or M; R127 replaced with H, or K; V129replaced with A, G, I, L, S, T, or M; V130 replaced with A, G, I, L, S,T, or M; Q131 replaced with N; V132 replaced with A, G, I, L, S, T, orM; F133 replaced with W, or Y; I134 replaced with A, G, L, S, T, M, orV; A135 replaced with G, I, L, S, T, M, or V; V136 replaced with A, G,I, L, S, T, or M; E137 replaced with D; R138 replaced with H, or K; D139replaced with E; A140 replaced with G, I, L, S, T, M, or V; Q141replaced with N; S143 replaced with A, G, I, L, T, M, or V; K144replaced with H, or R; T145 replaced with A, G, I, L, S, M, or V; Q147replaced with N; V148 replaced with A, G, I, L, S, T, or M; F149replaced with W, or Y; L150 replaced with A, G, I, S, T, M, or V; S151replaced with A, G, I, L, T, M, or V; W153 replaced with F, or Y; A154replaced with G, I, L, S, T, M, or V; N156 replaced with Q; I157replaced with A, G, L, S, T, M, or V; S158 replaced with A, G, I, L, T,M, or V; E159 replaced with D; I160 replaced with A, G, L, S, T, M, orV; T161 replaced with A, G, I, L, S, M, or V; Y162 replaced with F, orW; S163 replaced with A, G, I, L, T, M, or V; W164 replaced with F, orY; R165 replaced with H, or K; R166 replaced with H, or K; E167 replacedwith D; T168 replaced with A, G, I, L, S, M, or V; T169 replaced with A,G, I, L, S, M, or V; M170 replaced with A, G, I, L, S, T, or V; D171replaced with E; F172 replaced with W, or Y; G173 replaced with A, I, L,S, T, M, or V; M174 replaced with A, G, I, L, S, T, or V; E175 replacedwith D; H177 replaced with K, or R; S178 replaced with A, G, I, L, T, M,or V; L179 replaced with A, G, I, S, T, M, or V; F180 replaced with W,or Y; T181 replaced with A, G, I, L, S, M, or V; D182 replaced with E;G183 replaced with A, I, L, S, T, M, or V; Q184 replaced with N; V185replaced with A, G, I, L, S, T, or M; L186 replaced with A, G, I, S, T,M, or V; S187 replaced with A, G, I, L, T, M, or V; I188 replaced withA, G, L, S, T, M, or V; S189 replaced with A, G, I, L, T, M, or V; L190replaced with A, G, I, S, T, M, or V; G191 replaced with A, I, L, S, T,M, or V; G193 replaced with A, I, L, S, T, M, or V; D194 replaced withE; R195 replaced with H, or K; D196 replaced with E; V197 replaced withA, G, I, L, S, T, or M; A198 replaced with G, I, L, S, T, M, or V; Y199replaced with F, or W; S200 replaced with A, G, I, L, T, M, or V; I202replaced with A, G, L, S, T, M, or V; V203 replaced with A, G, I, L, S,T, or M; S204 replaced with A, G, I, L, T, M, or V; N205 replaced withQ; V207 replaced with A, G, I, L, S, T, or M; S208 replaced with A, G,I, L, T, M, or V; W209 replaced with F, or Y; D210 replaced with E; L211replaced with A, G, I, S, T, M, or V; A212 replaced with G, I, L, S, T,M, or V; T213 replaced with A, G, I, L, S, M, or V; V214 replaced withA, G, I, L, S, T, or M; T215 replaced with A, G, I, L, S, M, or V; W217replaced with F, or Y; D218 replaced with E; S219 replaced with A, G, I,L, T, M, or V; H221 replaced with K, or R; H222 replaced with K, or R;E223 replaced with D; A224 replaced with G, I, L, S, T, M, or V; A225replaced with G, I, L, S, T, M, or V; G227 replaced with A, I, L, S, T,M, or V; K228 replaced with H, or R; A229 replaced with G, I, L, S, T,M, or V; S230 replaced with A, G, I, L, T, M, or V; Y231 replaced withF, or W; K232 replaced with H, or R; D233 replaced with E; V234 replacedwith A, G, I, L, S, T, or M; L235 replaced with A, G, I, S, T, M, or V;L236 replaced with A, G, I, S, T, M, or V; V237 replaced with A, G, I,L, S, T, or M; V238 replaced with A, G, I, L, S, T, or M; V239 replacedwith A, G, I, L, S, T, or M; V241 replaced with A, G, I, L, S, T, or M;S242 replaced with A, G, I, L, T, M, or V; L243 replaced with A, G, I,S, T, M, or V; L244 replaced with A, G, I, S, T, M, or V; L245 replacedwith A, G, I, S, T, M, or V; M246 replaced with A, G, I, L, S, T, or V;L247 replaced with A, G, I, S, T, M, or V; V248 replaced with A, G, I,L, S, T, or M; T249 replaced with A, G, I, L, S, M, or V; L250 replacedwith A, G, I, S, T, M, or V; F251 replaced with W, or Y; S252 replacedwith A, G, I, L, T, M, or V; A253 replaced with G, I, L, S, T, M, or V;W254 replaced with F, or Y; H255 replaced with K, or R; W256 replacedwith F, or Y; S260 replaced with A, G, I, L, T, M, or V; G261 replacedwith A, I, L, S, T, M, or V; K262 replaced with H, or R; K263 replacedwith H, or R; K264 replaced with H, or R; K265 replaced with H, or R;D266 replaced with E; V267 replaced with A, G, I, L, S, T, or M; H268replaced with K, or R; A269 replaced with G, I, L, S, T, M, or V; D270replaced with E; R271 replaced with H, or K; V272 replaced with A, G, I,L, S, T, or M; G273 replaced with A, I, L, S, T, M, or V; E275 replacedwith D; T276 replaced with A, G, I, L, S, M, or V; E277 replaced with D;N278 replaced with Q; L280 replaced with A, G, I, S, T, M, or V; V281replaced with A, G, I, L, S, T, or M; Q282 replaced with N; D283replaced with E; L284 replaced with A, G, I, S, T, M, or V.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have an increased D-SLAM activityor function, while the remaining D-SLAM activities or functions aremaintained. More preferably, the resulting constructs have more than oneincreased D-SLAM activity or function, while the remaining D-SLAMactivities or functions are maintained.

Besides conservative amino acid substitution, variants of D-SLAM include(i) substitutions with one or more of the non-conserved amino acidresidues, where the substituted amino acid residues may or may not beone encoded by the genetic code, or (ii) substitution with one or moreof amino acid residues having a substituent group, or (iii) fusion ofthe mature polypeptide with another compound, such as a compound toincrease the stability and/or solubility of the polypeptide (forexample, polyethylene glycol), or (iv) fusion of the polypeptide withadditional amino acids, such as an IgG Fc fusion region peptide, orleader or secretory sequence, or a sequence facilitating purification.Such variant polypeptides are deemed to be within the scope of thoseskilled in the art from the teachings herein.

For example, D-SLAM polypeptide variants containing amino acidsubstitutions of charged amino acids with other charged or neutral aminoacids may produce proteins with improved characteristics, such as lessaggregation. Aggregation of pharmaceutical formulations both reducesactivity and increases clearance due to the aggregate's immunogenicactivity. (Pinckard et al., Clin. Exp. Immunol. 2:331-340 (1967);Robbins et al., Diabetes 36: 838-845 (1987); Cleland et al., Crit. Rev.Therapeutic Drug Carrier Systems 10:307-377 (1993).)

For example, preferred non-conservative substitutions of D-SLAM include:M1 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V2 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; M3 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; R4 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; P5 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; L6 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; W7 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; S8 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L9 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L10 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L11 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; W12 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; E13 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; A14 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; L15 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; L16 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P17 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; I18replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; T19 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V20 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; T21 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G22 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A23replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q24 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; V25 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L26 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; S27 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; K28 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; V29 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G30 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G31 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; S32 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; V33 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L34 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L35 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V36 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A37 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; A38 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; R39 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; P40 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or C; P41 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, N, Q, F, W, Y, or C; G42 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; F43 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M,V, P, or C; Q44 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; V45 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;R46 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;E47 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; A48 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; I49 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; W50 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R51 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S52 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; L53 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; W54 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; P55 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, or C; S56 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; E57 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; E58 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; L59 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L60replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A61 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; T62 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; F63 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; F64 replaced with D, E, H, K, R, N, Q, A, G, I,L, S, T, M, V, P, or C; R65 replaced with D, E, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; G66 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; S67 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L68replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E69 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T70 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; L71 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; Y72 replaced with D, E, H, K, R, N, Q, A,G, I, L, S, T, M, V, P, or C; H73 replaced with D, E, A, G, I, L, S, T,M, V, N, Q, F, W, Y, P, or C; S74 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; R75 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; F76 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T,M, V, P, or C; L77 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G78 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; R79 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A80 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; Q81 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; L82 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; H83 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; S84 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; N85 replaced with D, E, H, K, R, A, G, I, L, S, T, M,V, F, W, Y, P, or C; L86 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; S87 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L88replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E89 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L90 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; G91 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P92 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; L93 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; E94 replaced with H, K, R, A, G, I, L, S, T, M, V,N, Q, F, W, Y, P, or C; S95 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; G96 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D97replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;S98 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G99 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; N100 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; F101 replaced with D, E,H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S102 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; V103 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; L104 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; M105 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V106replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D107 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T108 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; R109 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; G110 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; Q111 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, F, W, Y, P, or C; P112 replaced with D, E, H, K, R, A, G,I, L, S, T, M, V, N, Q, F, W, Y, or C; W113 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; T114 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; Q115 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, F, W, Y, P, or C; T116 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; L117 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;Q118 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, orC; L119 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K120replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V121replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y122 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D123 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A124 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V125 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P126 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; R127 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; P128 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, N, Q, F, W, Y, or C; V129 replaced with D, E, H, K,R, N, Q, F, W, Y, P, or C; V130 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; Q131 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; V132 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;F133 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C;I134 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A135 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; V136 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; E137 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; R138 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; D139 replaced with H, K, R, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; A140 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; Q141 replaced with D, E, H, K, R, A, G, I, L, S, T,M, V, F, W, Y, P, or C; P142 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or C; S143 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; K144 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F,W, Y, P, or C; T145 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;C146 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,or P; Q147 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y,P, or C; V148 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; F149replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; L150replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S151 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; C152 replaced with D, E, H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; W153 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; A154 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; P155 replaced with D, E, H, K, R, A, G, I,L, S, T, M, V, N, Q, F, W, Y, or C; N156 replaced with D, E, H, K, R, A,G, I, L, S, T, M, V, F, W, Y, P, or C; 1157 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; S158 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; E159 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; I160 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T161 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Y162 replacedwith D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; S163 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; W164 replaced with D, E, H,K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; R165 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R166 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E167 replaced with H, K, R,A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; T168 replaced with D, E,H, K, R, N, Q, F, W, Y, P, or C; T169 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; M170 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; D171 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; F172 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; G173 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; M174replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; E175 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; P176 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; H177replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; S178replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L179 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; F180 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; T181 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; D182 replaced with H, K, R, A, G, I, L, S, T, M,V, N, Q, F, W, Y, P, or C; G183 replaced with D, E, H, K, R, N, Q, F, W,Y, P, or C; Q184 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F,W, Y, P, or C; V185 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;L186 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S187 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; I188 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; S189 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; L190 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;G191 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P192 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G193replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; D194 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; R195 replacedwith D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D196 replacedwith H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V197replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A198 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; Y199 replaced with D, E, H, K, R,N, Q, A, G, I, L, S, T, M, V, P, or C; S200 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; C201 replaced with D, E, H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, or P; I202 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V203 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;S204 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; N205 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; P206replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; V207 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S208replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W209 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D210 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; L211 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; A212 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; T213 replaced with D, E, H, K, R, N, Q, F,W, Y, P, or C; V214 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;T215 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P216 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; W217replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; D218replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;S219 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; C220 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; H221replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; H222replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; E223replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;A224 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A225 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; P226 replaced with D, E, H,K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C; G227 replaced with D,E, H, K, R, N, Q, F, W, Y, P, or C; K228 replaced with D, E, A, G, I, L,S, T, M, V, N, Q, F, W, Y, P, or C; A229 replaced with D, E, H, K, R, N,Q, F, W, Y, P, or C; S230 replaced with D, E, H, K, R, N, Q, F, W, Y, P,or C; Y231 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P,or C; K232 replaced with D, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P,or C; D233 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y,P, or C; V234 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L235replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L236 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; V237 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; V238 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; V239 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P240replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; V241 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; S242replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L243 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; L244 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L245 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; M246 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; L247replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; V248 replaced withD, E, H, K, R, N, Q, F, W, Y, P, or C; T249 replaced with D, E, H, K, R,N, Q, F, W, Y, P, or C; L250 replaced with D, E, H, K, R, N, Q, F, W, Y,P, or C; F251 replaced with D, E, H, K, R, N, Q, A, G, I, L, S, T, M, V,P, or C; S252 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; A253replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; W254 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; H255 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; W256 replaced withD, E, H, K, R, N, Q, A, G, I, L, S, T, M, V, P, or C; C257 replaced withD, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or P; P258replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, orC; C259 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or P; S260 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; G261replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; K262 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K263 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K264 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; K265 replaced withD, E, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; D266 replaced withH, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; V267 replacedwith D, E, H, K, R, N, Q, F, W, Y, P, or C; H268 replaced with D, E, A,G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C; A269 replaced with D, E, H,K, R, N, Q, F, W, Y, P, or C; D270 replaced with H, K, R, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; R271 replaced with D, E, A, G, I, L, S,T, M, V, N, Q, F, W, Y, P, or C; V272 replaced with D, E, H, K, R, N, Q,F, W, Y, P, or C; G273 replaced with D, E, H, K, R, N, Q, F, W, Y, P, orC; P274 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, or C; E275 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W,Y, P, or C; T276 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;E277 replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, orC; N278 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P,or C; P279 replaced with D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F,W, Y, or C; L280 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C;V281 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; Q282 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, F, W, Y, P, or C; D283replaced with H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, P, or C;L284 replaced with D, E, H, K, R, N, Q, F, W, Y, P, or C; P285 replacedwith D, E, H, K, R, A, G, I, L, S, T, M, V, N, Q, F, W, Y, or C.

The resulting constructs can be routinely screened for activities orfunctions described throughout the specification and known in the art.Preferably, the resulting constructs have loss of a D-SLAM activity orfunction, while the remaining D-SLAM activities or functions aremaintained. More preferably, the resulting constructs have more than oneloss of D-SLAM activity or function, while the remaining D-SLAMactivities or functions are maintained.

Additionally, more than one amino acid (e.g., 2, 3, 4, 5, 6, 7, 8, 9 and10) can be replaced with the substituted amino acids as described above(either conservative or nonconservative).

A further embodiment of the invention relates to a polypeptide whichcomprises, or alternatively consists of, the amino acid sequence of aD-SLAM polypeptide having an amino acid sequence which contains at leastone amino acid substitution, but not more than 50 amino acidsubstitutions, even more preferably, not more than 40 amino acidsubstitutions, still more preferably, not more than 30 amino acidsubstitutions, and still even more preferably, not more than 20 aminoacid substitutions. Of course, in order of ever-increasing preference,it is highly preferable for a peptide or polypeptide to have an aminoacid sequence which comprises, or alternatively consists of, the aminoacid sequence of a D-SLAM polypeptide, which contains at least one, butnot more than 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid substitutions.In specific embodiments, the number of additions, substitutions, and/ordeletions in the amino acid sequence of FIGS. 1A-1D or fragments thereof(e.g., the mature form and/or other fragments described herein), is1-5,5-10, 5-25, 5-50, 10-50 or 50-150, conservative amino acidsubstitutions are preferable.

Polynucleotide and Polypeptide Fragments

The present invention is further directed to fragments of the isolatednucleic acid molecules described herein. By a fragment of an isolatednucleic acid molecule having, for example, the nucleotide sequence ofthe deposited cDNA (clone HDPJO39), a nucleotide sequence encoding thepolypeptide sequence encoded by the deposited cDNA, a nucleotidesequence encoding the polypeptide sequence depicted in FIG. 1 (SEQ IDNO:2), the nucleotide sequence shown in FIG. 1 (SEQ ID NO:1), or thecomplementary strand thereto, is intended fragments at least 15 nt, andmore preferably at least about 20 nt, still more preferably at least 30nt, and even more preferably, at least about 40, 50, 100, 150, 200, 250,300, 325, 350, 375, 400, 450, 500, 550, or 600 nt in length. Thesefragments have numerous uses that include, but are not limited to,diagnostic probes and primers as discussed herein. Of course, largerfragments, such as those of 501-1500 nt in length are also usefulaccording to the present invention as are fragments corresponding tomost, if not all, of the nucleotide sequences of the deposited cDNA(clone HDPJO39) or as shown in FIG. 1 (SEQ ID NO:1). By a fragment atleast 20 nt in length, for example, is intended fragments which include20 or more contiguous bases from, for example, the nucleotide sequenceof the deposited cDNA, or the nucleotide sequence as shown in FIG. 1(SEQ ID NO:1).

Moreover, representative examples of D-SLAM polynucleotide fragmentsinclude, for example, fragments having a sequence from about nucleotidenumber 1-50, 51-100, 101-150, 151-200, 201-250, 251-300, 301-350,351-400, 401-450, 451-500, 501-550, 551-600, 651-700, 701-750, 751-800,800-850, 851-900, 901-950, 951-1000, 1001-1050, 1051-1100, 1101-1150,1151-1200, 1201-1250, 1251-1300, 1301-1350, 1351-1400, 1401-1450,1451-1500, 1501-1550, 1551-1600, 1601-1650, 1651-1700, 1701-1750,1751-1800, 1801-1850, 1851-1900, 1901-1950, 1951-2000, and/or 2001 tothe end of SEQ ID NO:1 or the complementary strand thereto, or the cDNAcontained in the deposited clone. In this context “about” includes theparticularly recited ranges, larger or smaller by several (5, 4, 3, 2,or 1) nucleotides, at either terminus or at both termini.

Preferably, the polynucleotide fragments of the invention encode apolypeptide which demonstrates a D-SLAM functional activity. By “apolypeptide demonstrating a D-SLAM functional activity” is intendedpolypeptides capable of exhibiting activity similar, but not necessarilyidentical, to a known functional activity of the D-SLAM polypeptides ofthe present invention (e.g., complete (full-length) D-SLAM, matureD-SLAM and soluble D-SLAM (e.g., having sequences contained in theextracellular domain of D-SLAM) as measured, for example, in aparticular immunoassay or biological assay. Such functional activitiesinclude, but are not limited to, biological activity (e.g., ability tobind monocyte derived dendritic cells, ability to upregulate cellsurface markers indicative of dendritic cell maturation or activation(such as, for example, HLA-DR, CD54, CD86, CD83), ability to inhibitB-cell proliferation, ability to inhibit BLyS induced cell proliferativeactivity), antigenicity (ability to bind or compete with a D-SLAMpolypeptide for binding to an anti-D-SLAM antibody), immunogenicity(ability to generate antibody which binds to a D-SLAM polypeptide),ability to form multimers with D-SLAM polypeptides of the invention,ability to form heteromultimers, ability to bind to a receptor orligand).

The functional activity of D-SLAM polypeptides, and fragments, variantsderivatives, and analogs thereof, can be assayed by various methods.

For example, in one embodiment where one is assaying for the ability tobind or compete with full-length D-SLAM polypeptide for binding toanti-D-SLAM antibody, various immunoassays known in the art can be used,including but not limited to, competitive and non-competitive assaysystems using techniques such as radioimmunoassays, ELISA (enzyme linkedimmunosorbent assay), “sandwich” immunoassays, immunoradiometric assays,gel diffusion precipitation reactions, immunodiffusion assays, in situimmunoassays (using colloidal gold, enzyme or radioisotope labels, forexample), western blots, precipitation reactions, agglutination assays(e.g., gel agglutination assays, hemagglutination assays), complementfixation assays, immunofluorescence assays, protein A assays, andimmunoelectrophoresis assays, etc. In one embodiment, antibody bindingis detected by detecting a label on the primary antibody. In anotherembodiment, the primary antibody is detected by detecting binding of asecondary antibody or reagent to the primary antibody. In a furtherembodiment, the secondary antibody is labeled. Many means are known inthe art for detecting binding in an immunoassay and are within the scopeof the present invention.

In another embodiment, where a D-SLAM ligand is identified, or theability of a polypeptide fragment, variant or derivative of theinvention to multimerize is being evaluated, binding can be assayed,e.g., by means well-known in the art, such as, for example, reducing andnon-reducing gel chromatography, protein affinity chromatography, andaffinity blotting. See generally, Phizicky, E., et al., 1995, Microbiol.Rev. 59:94-123. In another embodiment, physiological correlates ofD-SLAM binding to its substrates (signal transduction) can be assayed.

In addition, assays described herein (see Examples) and otherwise knownin the art may routinely be applied to measure the ability of D-SLAMpolypeptides and fragments, variants derivatives and analogs thereof toelicit D-SLAM related biological activity (either in vitro or in vivo).Other methods will be known to the skilled artisan and are within thescope of the invention.

The present invention is further directed to fragments of the D-SLAMpolypeptide described herein. By a fragment of an isolated the D-SLAMpolypeptide, for example, encoded by the deposited cDNA (clone HDPJO39),the polypeptide sequence encoded by the deposited cDNA, the polypeptidesequence depicted in FIG. 1 (SEQ ID NO:2), is intended to encompasspolypeptide fragments contained in SEQ ID NO:2 or encoded by the cDNAcontained in the deposited clone. Protein fragments may be“free-standing,” or comprised within a larger polypeptide of which thefragment forms a part or region, most preferably as a single continuousregion. Representative examples of polypeptide fragments of theinvention, include, for example, fragments from about amino acid number1-20, 21-40, 41-60, 61-80, 81-100, 102-120, 121-140, 141-160, 161-180,181-200, 201-220, 221-240, 241-260, 261-280, or 281 to the end of thecoding region. Moreover, polypeptide fragments can be at least 20, 30,40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, or 150 amino acids inlength. In this context “about” includes the particularly recitedranges, larger or smaller by several (5, 4, 3, 2, or 1) amino acids, ateither extreme or at both extremes.

Even if deletion of one or more amino acids from the N-terminus of aprotein results in modification of loss of one or more biologicalfunctions of the protein, other functional activities (e.g., biologicalactivities, ability to multimerize, ability to bind D-SLAM ligand) maystill be retained. For example, the ability of shortened D-SLAM muteinsto induce and/or bind to antibodies which recognize the complete ormature forms of the polypeptides generally will be retained when lessthan the majority of the residues of the complete or mature polypeptideare removed from the N-terminus. Whether a particular polypeptidelacking N-terminal residues of a complete polypeptide retains suchimmunologic activities can readily be determined by routine methodsdescribed herein and otherwise known in the art. It is not unlikely thatan D-SLAM mutein with a large number of deleted N-terminal amino acidresidues may retain some biological or immunogenic activities. In fact,peptides composed of as few as six D-SLAM amino acid residues may oftenevoke an immune response.

Accordingly, polypeptide fragments include the secreted D-SLAM proteinas well as the mature form. Further preferred polypeptide fragmentsinclude the secreted D-SLAM protein or the mature form having acontinuous series of deleted residues from the amino or the carboxyterminus, or both. For example, any number of amino acids, ranging from1-60, can be deleted from the amino terminus of either the secretedD-SLAM polypeptide or the mature form. Similarly, any number of aminoacids, ranging from 1-30, can be deleted from the carboxy terminus ofthe secreted D-SLAM protein or mature form. Furthermore, any combinationof the above amino and carboxy terminus deletions are preferred.Similarly, polynucleotide fragments encoding these D-SLAM polypeptidefragments are also preferred. The present invention is also directed tonucleic acid molecules comprising, or alternatively, consisting of, apolynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%or 99% identical to the polynucleotide sequence encoding the D-SLAMpolypeptides described above. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence. Polypeptides encoded by these nucleic acids and/orpolynucleotide sequences are also encompassed by the invention, as arepolypeptides comprising, or alternatively consisting of, an amino acidsequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence described above, andpolynucleotides that encode such polypeptides.

Particularly, N-terminal deletions of the D-SLAM polypeptide can bedescribed by the general formula m-285, where m is an integer from 2 to280, where m corresponds to the position of the amino acid residueidentified in SEQ ID NO:2. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, an amino acid sequence selected from the group consistingof residues V-2 to P-285; M-3 to P-285; R-4 to P-285; P-5 to P-285; L-6to P-285; W-7 to P-285; S-8 to P-285; L-9 to P-285; L-10 to P-285; L-11to P-285; W-12 to P-285; E-13 to P-285; A-14 to P-285; L-15 to P-285;L-16 to P-285; P-17 to P-285; I-18 to P-285; T-19 to P-285; V-20 toP-285; T-21 to P-285; G-22 to P-285; A-23 to P-285; Q-24 to P-285; V-25to P-285; L-26 to P-285; S-27 to P-285; K-28 to P-285; V-29 to P-285;G-30 to P-285; G-31 to P-285; S-32 to P-285; V-33 to P-285; L-34 toP-285; L-35 to P-285; V-36 to P-285; A-37 to P-285; A-38 to P-285; R-39to P-285; P-40 to P-285; P-41 to P-285; G-42 to P-285; F-43 to P-285;Q-44 to P-285; V-45 to P-285; R-46 to P-285; E-47 to P-285; A-48 toP-285; I-49 to P-285; W-50 to P-285; R-51 to P-285; S-52 to P-285; L-53to P-285; W-54 to P-285; P-55 to P-285; S-56 to P-285; E-57 to P-285;E-58 to P-285; L-59 to P-285; L-60 to P-285; A-61 to P-285; T-62 toP-285; F-63 to P-285; F-64 to P-285; R-65 to P-285; G-66 to P-285; S-67to P-285; L-68 to P-285; E-69 to P-285; T-70 to P-285; L-71 to P-285;Y-72 to P-285; H-73 to P-285; S-74 to P-285; R-75 to P-285; F-76 toP-285; L-77 to P-285; G-78 to P-285; R-79 to P-285; A-80 to P-285; Q-81to P-285; L-82 to P-285; H-83 to P-285; S-84 to P-285; N-85 to P-285;L-86 to P-285; S-87 to P-285; L-88 to P-285; E-89 to P-285; L-90 toP-285; G-91 to P-285; P-92 to P-285; L-93 to P-285; E-94 to P-285; S-95to P-285; G-96 to P-285; D-97 to P-285; S-98 to P-285; G-99 to P-285;N-100 to P-285; F-101 to P-285; S-102 to P-285; V-103 to P-285; L-104 toP-285; M-105 to P-285; V-106 to P-285; D-107 to P-285; T-108 to P-285;R-109 to P-285; G-110 to P-285; Q-111 to P-285; P-112 to P-285; W-113 toP-285; T-114 to P-285; Q-115 to P-285; T-116 to P-285; L-117 to P-285;Q-118 to P-285; L-119 to P-285; K-120 to P-285; V-121 to P-285; Y-122 toP-285; D-123 to P-285; A-124 to P-285; V-125 to P-285; P-126 to P-285;R-127 to P-285; P-128 to P-285; V-129 to P-285; V-130 to P-285; Q-131 toP-285; V-132 to P-285; F-133 to P-285; I-134 to P-285; A-135 to P-285;V-136 to P-285; E-137 to P-285; R-138 to P-285; D-139 to P-285; A-140 toP-285; Q-141 to P-285; P-142 to P-285; S-143 to P-285; K-144 to P-285;T-145 to P-285; C-146 to P-285; Q-147 to P-285; V-148 to P-285; F-149 toP-285; L-150 to P-285; S-151 to P-285; C-152 to P-285; W-153 to P-285;A-154 to P-285; P-155 to P-285; N-156 to P-285; I-157 to P-285; S-158 toP-285; E-159 to P-285; I-160 to P-285; T-161 to P-285; Y-162 to P-285;S-163 to P-285; W-164 to P-285; R-165 to P-285; R-166 to P-285; E-167 toP-285; T-168 to P-285; T-169 to P-285; M-170 to P-285; D-171 to P-285;F-172 to P-285; G-173 to P-285; M-174 to P-285; E-175 to P-285; P-176 toP-285; H-177 to P-285; S-178 to P-285; L-179 to P-285; F-180 to P-285;T-181 to P-285; D-182 to P-285; G-183 to P-285; Q-184 to P-285; V-185 toP-285; L-186 to P-285; S-187 to P-285; I-188 to P-285; S-189 to P-285;L-190 to P-285; G-191 to P-285; P-192 to P-285; G-193 to P-285; D-194 toP-285; R-195 to P-285; D-196 to P-285; V-197 to P-285; A-198 to P-285;Y-199 to P-285; S-200 to P-285; C-201 to P-285; I-202 to P-285; V-203 toP-285; S-204 to P-285; N-205 to P-285; P-206 to P-285; V-207 to P-285;S-208 to P-285; W-209 to P-285; D-210 to P-285; L-211 to P-285; A-212 toP-285; T-213 to P-285; V-214 to P-285; T-215 to P-285; P-216 to P-285;W-217 to P-285; D-218 to P-285; S-219 to P-285; C-220 to P-285; H-221 toP-285; H-222 to P-285; E-223 to P-285; A-224 to P-285; A-225 to P-285;P-226 to P-285; G-227 to P-285; K-228 to P-285; A-229 to P-285; S-230 toP-285; Y-231 to P-285; K-232 to P-285; D-233 to P-285; V-234 to P-285;L-235 to P-285; L-236 to P-285; V-237 to P-285; V-238 to P-285; V-239 toP-285; P-240 to P-285; V-241 to P-285; S-242 to P-285; L-243 to P-285;L-244 to P-285; L-245 to P-285; M-246 to P-285; L-247 to P-285; V-248 toP-285; T-249 to P-285; L-250 to P-285; F-251 to P-285; S-252 to P-285;A-253 to P-285; W-254 to P-285; H-255 to P-285; W-256 to P-285; C-257 toP-285; P-258 to P-285; C-259 to P-285; S-260 to P-285; G-261 to P-285;K-262 to P-285; K-263 to P-285; K-264 to P-285; K-265 to P-285; D-266 toP-285; V-267 to P-285; H-268 to P-285; A-269 to P-285; D-270 to P-285;R-271 to P-285; V-272 to P-285; G-273 to P-285; P-274 to P-285; E-275 toP-285; T-276 to P-285; E-277 to P-285; N-278 to P-285; P-279 to P-285;and L-280 to P-285 of SEQ ID NO:2. Polypeptides encoded by thesepolynucleotides are also encompassed by the invention. The presentinvention is also directed to nucleic acid molecules comprising, oralternatively, consisting of, a polynucleotide sequence at least 80%,85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotidesequence encoding the D-SLAM polypeptides described above. The presentinvention also encompasses the above polynucleotide sequences fused to aheterologous polynucleotide sequence. Polypeptides encoded by thesenucleic acids and/or polynucleotide sequences are also encompassed bythe invention, as are polypeptides comprising, or alternativelyconsisting of, an amino acid sequence at least 80%, 85%, 90%, 92%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence describedabove, and polynucleotides that encode such polypeptides.

Also as mentioned above, even if deletion of one or more amino acidsfrom the C-terminus of a protein results in modification of loss of oneor more biological functions of the protein, other functional activities(e.g., biological activities, ability to multimerize, ability to bindD-SLAM ligand) may still be retained. For example the ability of theshortened D-SLAM mutein to induce and/or bind to antibodies whichrecognize the complete or mature forms of the polypeptide generally willbe retained when less than the majority of the residues of the completeor mature polypeptide are removed from the C-terminus. Whether aparticular polypeptide lacking C-terminal residues of a completepolypeptide retains such immunologic activities can readily bedetermined by routine methods described herein and otherwise known inthe art. It is not unlikely that an D-SLAM mutein with a large number ofdeleted C-terminal amino acid residues may retain some biological orimmunogenic activities. In fact, peptides composed of as few as sixD-SLAM amino acid residues may often evoke an immune response.

Accordingly, the present invention further provides polypeptides havingone or more residues deleted from the carboxy terminus of the amino acidsequence of the D-SLAM polypeptide shown in FIG. 1 (SEQ ID NO:2), asdescribed by the general formula 1-n, where n is an integer from 7 to284, where n corresponds to the position of amino acid residueidentified in SEQ ID NO:2. More in particular, the invention providespolynucleotides encoding polypeptides comprising, or alternativelyconsisting of, an amino acid sequence selected from the group consistingof residues M-1 to L-284; M-1 to D-283; M-1 to Q-282; M-1 to V-281; M-1to L-280; M-1 to P-279; M-1 to N-278; M-1 to E-277; M-1 to T-276; M-1 toE-275; M-1 to P-274; M-1 to G-273; M-1 to V-272; M-1 to R-271; M-1 toD-270; M-1 to A-269; M-1 to H-268; M-1 to V-267; M-1 to D-266; M-1 toK-265; M-1 to K-264; M-1 to K-263; M-1 to K-262; M-1 to G-261; M-1 toS-260; M-1 to C-259; M-1 to P-258; M-1 to C-257; M-1 to W-256; M-1 toH-255; M-1 to W-254; M-1 to A-253; M-1 to S-252; M-1 to F-251; M-1 toL-250; M-1 to T-249; M-1 to V-248; M-1 to L-247; M-1 to M-246; M-1 toL-245; M-1 to L-244; M-1 to L-243; M-1 to S-242; M-1 to V-241; M-1 toP-240; M-1 to V-239; M-1 to V-238; M-1 to V-237; M-1 to L-236; M-1 toL-235; M-1 to V-234; M-1 to D-233; M-1 to K-232; M-1 to Y-231; M-1 toS-230; M-1 to A-229; M-1 to K-228; M-1 to G-227; M-1 to P-226; M-1 toA-225; M-1 to A-224; M-1 to E-223; M-1 to H-222; M-1 to H-221; M-1 toC-220; M-1 to S-219; M-1 to D-218; M-1 to W-217; M-1 to P-216; M-1 toT-215; M-1 to V-214; M-1 to T-213; M-1 to A-212; M-1 to L-211; M-1 toD-210; M-1 to W-209; M-1 to S-208; M-1 to V-207; M-1 to P-206; M-1 toN-205; M-1 to S-204; M-1 to V-203; M-1 to I-202; M-1 to C-201; M-1 toS-200; M-1 to Y-199; M-1 to A-198; M-1 to V-197; M-1 to D-196; M-1 toR-195; M-1 to D-194; M-1 to G-193; M-1 to P-192; M-1 to G-191; M-1 toL-190; M-1 to S-189; M-1 to I-188; M-1 to S-187; M-1 to L-186; M-1 toV-185; M-1 to Q-184; M-1 to G-183; M-1 to D-182; M-1 to T-181; M-1 toF-180; M-1 to L-179; M-1 to S-178; M-1 to H-177; M-1 to P-176; M-1 toE-175; M-1 to M-174; M-1 to G-173; M-1 to F-172; M-1 to D-171; M-1 toM-170; M-1 to T-169; M-1 to T-168; M-1 to E-167; M-1 to R-166; M-1 toR-165; M-1 to W-164; M-1 to S-163; M-1 to Y-162; M-1 to T-161; M-1 toI-160; M-1 to E-159; M-1 to S-158; M-1 to I-157; M-1 to N-156; M-1 toP-155; M-1 to A-154; M-1 to W-153; M-1 to C-152; M-1 to S-151; M-1 toL-150; M-1 to F-149; M-1 to V-148; M-1 to Q-147; M-1 to C-146; M-1 toT-145; M-1 to K-144; M-1 to S-143; M-1 to P-142; M-1 to Q-141; M-1 toA-140; M-1 to D-139; M-1 to R-138; M-1 to E-137; M-1 to V-136; M-1 toA-135; M-1 to I-134; M-1 to F-133; M-1 to V-132; M-1 to Q-131; M-1 toV-130; M-1 to V-129; M-1 to P-128; M-1 to R-127; M-1 to P-126; M-1 toV-125; M-1 to A-124; M-1 to D-123; M-1 to Y-122; M-1 to V-121; M-1 toK-120; M-1 to L-119; M-1 to Q-118; M-1 to L-117; M-1 to T-116; M-1 toQ-115; M-1 to T-114; M-1 to W-113; M-1 to P-112; M-1 to Q-111; M-1 toG-110; M-1 to R-109; M-1 to T-108; M-1 to D-107; M-1 to V-106; M-1 toM-105; M-1 to L-104; M-1 to V-103; M-1 to S-102; M-1 to F-101; M-1 toN-100; M-1 to G-99; M-1 to S-98; M-1 to D-97; M-1 to G-96; M-1 to S-95;M-1 to E-94; M-1 to L-93; M-1 to P-92; M-1 to G-91; M-1 to L-90; M-1 toE-89; M-1 to L-88; M-1 to S-87; M-1 to L-86; M-1 to N-85; M-1 to S-84;M-1 to H-83; M-1 to L-82; M-1 to Q-81; M-1 to A-80; M-1 to R-79; M-1 toG-78; M-1 to L-77; M-1 to F-76; M-1 to R-75; M-1 to S-74; M-1 to H-73;M-1 to Y-72; M-1 to L-71; M-1 to T-70; M-1 to E-69; M-1 to L-68; M-1 toS-67; M-1 to G-66; M-1 to R-65; M-1 to F-64; M-1 to F-63; M-1 to T-62;M-1 to A-61; M-1 to L-60; M-1 to L-59; M-1 to E-58; M-1 to E-57; M-1 toS-56; M-1 to P-55; M-1 to W-54; M-1 to L-53; M-1 to S-52; M-1 to R-51;M-1 to W-50; M-1 to I-49; M-1 to A-48; M-1 to E-47; M-1 to R-46; M-1 toV-45; M-1 to Q-44; M-1 to F-43; M-1 to G-42; M-1 to P-41; M-1 to P-40;M-1 to R-39; M-1 to A-38; M-1 to A-37; M-1 to V-36; M-1 to L-35; M-1 toL-34; M-1 to V-33; M-1 to S-32; M-1 to G-31; M-1 to G-30; M-1 to V-29;M-1 to K-28; M-1 to S-27; M-1 to L-26; M-1 to V-25; M-1 to Q-24; M-1 toA-23; M-1 to G-22; M-1 to T-21; M-1 to V-20; M-1 to T-19; M-1 to I-18;M-1 to P-17; M-1 to L-16; M-1 to L-15; M-1 to A-14; M-1 to E-13; M-1 toW-12; M-1 to L-11; M-1 to L-10; M-1 to L-9; M-1 to S-8; and M-1 to W-7of SEQ ID NO:2. Polypeptides encoded by these polynucleotides are alsoencompassed by the invention. The present invention is also directed tonucleic acid molecules comprising, or alternatively, consisting of, apolynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%or 99% identical to the polynucleotide sequence encoding the D-SLAMpolypeptides described above. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence. Polypeptides encoded by these nucleic acids and/orpolynucleotide sequences are also encompassed by the invention, as arepolypeptides comprising, or alternatively consisting of, an amino acidsequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence described above, andpolynucleotides that encode such polypeptides.

In addition, any of the above listed N- or C-terminal deletions can becombined to produce a N- and C-terminal deleted D-SLAM polypeptide. Theinvention also provides polypeptides having one or more amino acidsdeleted from both the amino and the carboxyl termini, which may bedescribed generally as having residues m-n of SEQ ID NO:2, where n and mare integers as described above. Polynucleotides encoding thesepolypeptides are also encompassed by the invention.

Moreover, preferred N- and C-terminal deletion mutants comprise, or inthe alternative consist of, the predicted secreted form of D-SLAM.Preferred secreted forms of the D-SLAM include polypeptides comprising,or alternatively consisting of, an amino acid sequence selected from thegroup consisting of residues M-1 to K-232; V-2 to K-232; M-3 to K-232;R-4 to K-232; P-5 to K-232; L-6 to K-232; W-7 to K-232; S-8 to K-232;L-9 to K-232; L-10 to K-232; L-11 to K-232; W-12 to K-232; E-13 toK-232; A-14 to K-232; L-15 to K-232; L-16 to K-232; P-17 to K-232; I-18to K-232; T-19 to K-232; V-20 to K-232; T-21 to K-232; G-22 to K-232;A-23 to K-232; Q-24 to K-232; V-25 to K-232; L-26 to K-232; S-27 toK-232; K-28 to K-232; V-29 to K-232; G-30 to K-232; G-31 to K-232; S-32to K-232; V-33 to K-232; L-34 to K-232; L-35 to K-232; V-36 to K-232;A-37 to K-232; A-38 to K-232; R-39 to K-232; P-40 to K-232; P-41 toK-232; G-42 to K-232; F-43 to K-232; Q-44 to K-232; V-45 to K-232; R-46to K-232; E-47 to K-232; A-48 to K-232; I-49 to K-232; W-50 to K-232;R-51 to K-232; S-52 to K-232; L-53 to K-232; W-54 to K-232; P-55 toK-232; S-56 to K-232; E-57 to K-232; E-58 to K-232; L-59 to K-232; L-60to K-232; A-61 to K-232; T-62 to K-232; F-63 to K-232; F-64 to K-232;R-65 to K-232; G-66 to K-232; S-67 to K-232; L-68 to K-232; E-69 toK-232; T-70 to K-232; L-71 to K-232; Y-72 to K-232; H-73 to K-232; S-74to K-232; R-75 to K-232; F-76 to K-232; L-77 to K-232; G-78 to K-232;R-79 to K-232; A-80 to K-232; Q-81 to K-232; L-82 to K-232; H-83 toK-232; S-84 to K-232; N-85 to K-232; L-86 to K-232; S-87 to K-232; L-88to K-232; E-89 to K-232; L-90 to K-232; G-91 to K-232; P-92 to K-232;L-93 to K-232; E-94 to K-232; S-95 to K-232; G-96 to K-232; D-97 toK-232; S-98 to K-232; G-99 to K-232; N-100 to K-232; F-101 to K-232;S-102 to K-232; V-103 to K-232; L-104 to K-232; M-105 to K-232; V-106 toK-232; D-107 to K-232; T-108 to K-232; R-109 to K-232; G-110 to K-232;Q-111 to K-232; P-112 to K-232; W-113 to K-232; T-114 to K-232; Q-115 toK-232; T-116 to K-232; L-117 to K-232; Q-118 to K-232; L-19 to K-232;K-120 to K-232; V-121 to K-232; Y-122 to K-232; D-123 to K-232; A-124 toK-232; V-125 to K-232; P-126 to K-232; R-127 to K-232; P-128 to K-232;V-129 to K-232; V-130 to K-232; Q-131 to K-232; V-132 to K-232; F-133 toK-232; I-134 to K-232; A-135 to K-232; V-136 to K-232; E-137 to K-232;R-138 to K-232; D-139 to K-232; A-140 to K-232; Q-141 to K-232; P-142 toK-232; S-143 to K-232; K-144 to K-232; T-145 to K-232; C-146 to K-232;Q-147 to K-232; V-148 to K-232; F-149 to K-232; L-150 to K-232; S-151 toK-232; C-152 to K-232; W-153 to K-232; A-154 to K-232; P-155 to K-232;N-156 to K-232; I-157 to K-232; S-158 to K-232; E-159 to K-232; I-160 toK-232; T-161 to K-232; Y-162 to K-232; S-163 to K-232; W-164 to K-232;R-165 to K-232; R-166 to K-232; E-167 to K-232; T-168 to K-232; T-169 toK-232; M-170 to K-232; D-171 to K-232; F-172 to K-232; G-173 to K-232;M-174 to K-232; E-175 to K-232; P-176 to K-232; H-177 to K-232; S-178 toK-232; L-179 to K-232; F-180 to K-232; T-181 to K-232; D-182 to K-232;G-183 to K-232; Q-184 to K-232; V-185 to K-232; L-186 to K-232; S-187 toK-232; I-188 to K-232; S-189 to K-232; L-190 to K-232; G-191 to K-232;P-192 to K-232; G-193 to K-232; D-194 to K-232; R-195 to K-232; D-196 toK-232; V-197 to K-232; A-198 to K-232; Y-199 to K-232; S-200 to K-232;C-201 to K-232; I-202 to K-232; V-203 to K-232; S-204 to K-232; N-205 toK-232; P-206 to K-232; V-207 to K-232; S-208 to K-232; W-209 to K-232;D-210 to K-232; L-211 to K-232; A-212 to K-232; T-213 to K-232; V-214 toK-232; T-215 to K-232; P-216 to K-232; W-217 to K-232; D-218 to K-232;S-219 to K-232; C-220 to K-232; H-221 to K-232; H-222 to K-232; E-223 toK-232; A-224 to K-232; A-225 to K-232; P-226 to K-232; and G-227 toK-232 of SEQ ID NO:2. Polypeptides encoded by these polynucleotides arealso encompassed by the invention. The present invention is alsodirected to nucleic acid molecules comprising, or alternatively,consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%,95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequenceencoding the D-SLAM polypeptides described above. The present inventionalso encompasses the above polynucleotide sequences fused to aheterologous polynucleotide sequence. Polypeptides encoded by thesenucleic acids and/or polynucleotide sequences are also encompassed bythe invention, as are polypeptides comprising, or alternativelyconsisting of, an amino acid sequence at least 80%, 85%, 90%, 92%, 95%,96%, 97%, 98% or 99% identical to the amino acid sequence describedabove, and polynucleotides that encode such polypeptides.

Additionally, preferred N- and C-terminal deletion mutants comprise, orin the alternative consist of, fragments lacking the predicted signalsequence of D-SLAM. Preferred fragments of D-SLAM include polypeptidescomprising, or alternatively consisting of, an amino acid sequenceselected from the group consisting of residues A-23 to L-284; A-23 toD-283; A-23 to Q-282; A-23 to V-281; A-23 to L-280; A-23 to P-279; A-23to N-278; A-23 to E-277; A-23 to T-276; A-23 to E-275; A-23 to P-274;A-23 to G-273; A-23 to V-272; A-23 to R-271; A-23 to D-270; A-23 toA-269; A-23 to H-268; A-23 to V-267; A-23 to D-266; A-23 to K-265; A-23to K-264; A-23 to K-263; A-23 to K-262; A-23 to G-261; A-23 to S-260;A-23 to C-259; A-23 to P-258; A-23 to C-257; A-23 to W-256; A-23 toH-255; A-23 to W-254; A-23 to A-253; A-23 to S-252; A-23 to F-251; A-23to L-250; A-23 to T-249; A-23 to V-248; A-23 to L-247; A-23 to M-246;A-23 to L-245; A-23 to L-244; A-23 to L-243; A-23 to S-242; A-23 toV-241; A-23 to P-240; A-23 to V-239; A-23 to V-238; A-23 to V-237; A-23to L-236; A-23 to L-235; A-23 to V-234; A-23 to D-233; A-23 to K-232;A-23 to Y-231; A-23 to S-230; A-23 to A-229; A-23 to K-228; A-23 toG-227; A-23 to P-226; A-23 to A-225; A-23 to A-224; A-23 to E-223; A-23to H-222; A-23 to H-221; A-23 to C-220; A-23 to S-219; A-23 to D-218;A-23 to W-217; A-23 to P-216; A-23 to T-215; A-23 to V-214; A-23 toT-213; A-23 to A-212; A-23 to L-211; A-23 to D-210; A-23 to W-209; A-23to S-208; A-23 to V-207; A-23 to P-206; A-23 to N-205; A-23 to S-204;A-23 to V-203; A-23 to I-202; A-23 to C-201; A-23 to S-200; A-23 toY-199; A-23 to A-198; A-23 to V-197; A-23 to D-196; A-23 to R-195; A-23to D-194; A-23 to G-193; A-23 to P-192; A-23 to G-191; A-23 to L-190;A-23 to S-189; A-23 to I-188; A-23 to S-187; A-23 to L-186; A-23 toV-185; A-23 to Q-184; A-23 to G-183; A-23 to D-182; A-23 to T-181; A-23to F-180; A-23 to L-179; A-23 to S-178; A-23 to H-177; A-23 to P-176;A-23 to E-175; A-23 to M-174; A-23 to G-173; A-23 to F-172; A-23 toD-171; A-23 to M-170; A-23 to T-169; A-23 to T-168; A-23 to E-167; A-23to R-166; A-23 to R-165; A-23 to W-164; A-23 to S-163; A-23 to Y-162;A-23 to T-161; A-23 to I-160; A-23 to E-159; A-23 to S-158; A-23 toI-157; A-23 to N-156; A-23 to P-155; A-23 to A-154; A-23 to W-153; A-23to C-152; A-23 to S-151; A-23 to L-150; A-23 to F-149; A-23 to V-148;A-23 to Q-147; A-23 to C-146; A-23 to T-145; A-23 to K-144; A-23 toS-143; A-23 to P-142; A-23 to Q-141; A-23 to A-140; A-23 to D-139; A-23to R-138; A-23 to E-137; A-23 to V-136; A-23 to A-135; A-23 to I-134;A-23 to F-133; A-23 to V-132; A-23 to Q-131; A-23 to V-130; A-23 toV-129; A-23 to P-128; A-23 to R-127; A-23 to P-126; A-23 to V-125; A-23to A-124; A-23 to D-123; A-23 to Y-122; A-23 to V-121; A-23 to K-120;A-23 to L-119; A-23 to Q-118; A-23 to L-117; A-23 to T-116; A-23 toQ-115; A-23 to T-114; A-23 to W-113; A-23 to P-112; A-23 to Q-111; A-23to G-110; A-23 to R-109; A-23 to T-108; A-23 to D-107; A-23 to V-106;A-23 to M-105; A-23 to L-104; A-23 to V-103; A-23 to S-102; A-23 toF-101; A-23 to N-100; A-23 to G-99; A-23 to S-98; A-23 to D-97; A-23 toG-96; A-23 to S-95; A-23 to E-94; A-23 to L-93; A-23 to P-92; A-23 toG-91; A-23 to L-90; A-23 to E-89; A-23 to L-88; A-23 to S-87; A-23 toL-86; A-23 to N-85; A-23 to S-84; A-23 to H-83; A-23 to L-82; A-23 toQ-81; A-23 to A-80; A-23 to R-79; A-23 to G-78; A-23 to L-77; A-23 toF-76; A-23 to R-75; A-23 to S-74; A-23 to H-73; A-23 to Y-72; A-23 toL-71; A-23 to T-70; A-23 to E-69; A-23 to L-68; A-23 to S-67; A-23 toG-66; A-23 to R-65; A-23 to F-64; A-23 to F-63; A-23 to T-62; A-23 toA-61; A-23 to L-60; A-23 to L-59; A-23 to E-58; A-23 to E-57; A-23 toS-56; A-23 to P-55; A-23 to W-54; A-23 to L-53; A-23 to S-52; A-23 toR-51; A-23 to W-50; A-23 to I-49; A-23 to A-48; A-23 to E-47; A-23 toR-46; A-23 to V-45; A-23 to Q-44; A-23 to F-43; A-23 to G-42; A-23 toP-41; A-23 to P-40; A-23 to R-39; A-23 to A-38; A-23 to A-37; A-23 toV-36; A-23 to L-35; A-23 to L-34; A-23 to V-33; A-23 to S-32; A-23 toG-31; A-23 to G-30; and A-23 to V-29 of SEQ ID NO:2. Polypeptidesencoded by these polynucleotides are also encompassed by the invention.The present invention is also directed to nucleic acid moleculescomprising, or alternatively, consisting of, a polynucleotide sequenceat least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to thepolynucleotide sequence encoding the D-SLAM polypeptides describedabove. The present invention also encompasses the above polynucleotidesequences fused to a heterologous polynucleotide sequence. Polypeptidesencoded by these nucleic acids and/or polynucleotide sequences are alsoencompassed by the invention, as are polypeptides comprising, oralternatively consisting of, an amino acid sequence at least 80%, 85%,90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the amino acid sequencedescribed above, and polynucleotides that encode such polypeptides.

The present application is also directed to proteins containingpolypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to the D-SLAM polypeptide sequence set forth herein m-n. Inpreferred embodiments, the application is directed to proteinscontaining polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%or 99% identical to polypeptides having the amino acid sequence of thespecific D-SLAM N- and C-terminal deletions recited herein.Polynucleotides encoded by these polypeptides are also encompassed bythe invention. The present invention is also directed to nucleic acidmolecules comprising, or alternatively, consisting of, a polynucleotidesequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to the polynucleotide sequence encoding the D-SLAMpolypeptides described above. The present invention also encompasses theabove polynucleotide sequences fused to a heterologous polynucleotidesequence. Polypeptides encoded by these nucleic acids and/orpolynucleotide sequences are also encompassed by the invention, as arepolypeptides comprising, or alternatively consisting of, an amino acidsequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%identical to the amino acid sequence described above, andpolynucleotides that encode such polypeptides.

Among the especially preferred fragments of the invention are fragmentscharacterized by structural or functional attributes of D-SLAM. Suchfragments include amino acid residues that comprise, or alternativelyconsist of, alpha-helix and alpha-helix forming regions(“alpha-regions”), beta-sheet and beta-sheet-forming regions(“beta-regions”), turn and turn-forming regions (“turn-regions”), coiland coil-forming regions (“coil-regions”), hydrophilic regions,hydrophobic regions, alpha amphipathic regions, beta amphipathicregions, surface forming regions, and high antigenic index regions(i.e., containing four or more contiguous amino acids having anantigenic index of greater than or equal to 1.5, as identified using thedefault parameters of the Jameson-Wolf program) of complete (i.e.,full-length) D-SLAM (SEQ ID NO:2). Certain preferred regions are thoseset out in FIG. 3 and include, but are not limited to, regions of theaforementioned types identified by analysis of the amino acid sequencedepicted in FIG. 1 (SEQ ID NO:2), such preferred regions include;Garnier-Robson predicted alpha-regions, beta-regions, turn-regions, andcoil-regions; Chou-Fasman predicted alpha-regions, beta-regions,turn-regions, and coil-regions; Kyte-Doolittle predicted hydrophilic andhydrophobic regions; Eisenberg alpha and beta amphipathic regions; Eminisurface-forming regions; and Jameson-Wolf high antigenic index regions,as predicted using the default parameters of these computer programs.Polynucleotides encoding these polypeptides are also encompassed by theinvention.

In additional embodiments, the polynucleotides of the invention encodefunctional attributes of D-SLAM. Preferred embodiments of the inventionin this regard include fragments that comprise, or alternatively consistof, alpha-helix and alpha-helix forming regions (“alpha-regions”),beta-sheet and beta-sheet forming regions (“beta-regions”), turn andturn-forming regions (“turn-regions”), coil and coil-forming regions(“coil-regions”), hydrophilic regions, hydrophobic regions, alphaamphipathic regions, beta amphipathic regions, flexible regions,surface-forming regions and high antigenic index regions of D-SLAM.

The data representing the structural or functional attributes of D-SLAMset forth in FIG. 1 and/or Table I, as described above, was generatedusing the various modules and algorithms of the DNA*STAR set on defaultparameters. In a preferred embodiment, the data presented in columnsVIII, IX, XIII, and XIV of Table I can be used to determine regions ofD-SLAM which exhibit a high degree of potential for antigenicity.Regions of high antigenicity are determined from the data presented incolumns VIII, IX, XIII, and/or IV by choosing values which representregions of the polypeptide which are likely to be exposed on the surfaceof the polypeptide in an environment in which antigen recognition mayoccur in the process of initiation of an immune response.

Certain preferred regions in these regards are set out in FIG. 3, butmay, as shown in Table I, be represented or identified by using tabularrepresentations of the data presented in FIG. 3. The DNA*STAR computeralgorithm used to generate FIG. 3 (set on the original defaultparameters) was used to present the data in FIG. 3 in a tabular format(See Table I). The tabular format of the data in FIG. 3 may be used toeasily determine specific boundaries of a preferred region.

The above-mentioned preferred regions set out in FIG. 3 and in Table Iinclude, but are not limited to, regions of the aforementioned typesidentified by analysis of the amino acid sequence set out in FIG. 1. Asset out in FIG. 3 and in Table I, such preferred regions includeGarnier-Robson alpha-regions, beta-regions, turn-regions, andcoil-regions, Chou-Fasman alpha-regions, beta-regions, and coil-regions,Kyte-Doolittle hydrophilic regions and hydrophobic regions, Eisenbergalpha- and beta-amphipathic regions, Karplus-Schulz flexible regions,Emini surface-forming regions and Jameson-Wolf regions of high antigenicindex.

Among highly preferred fragments in this regard are those that comprise,or alternatively consist of, regions of D-SLAM that combine severalstructural features, such as several of the features set out in Table 1.

Other preferred fragments are biologically active D-SLAM fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the D-SLAM polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

However, many polynucleotide sequences, such as EST sequences, arepublicly available and accessible through sequence databases. Some ofthese sequences are related to SEQ ID NO:1 and may have been publiclyavailable prior to conception of the present invention. Preferably, suchrelated polynucleotides are specifically excluded from the scope of thepresent invention. For example, the following ESTs are preferablyexcluded from the present invention: AA917335; AI094818, AI298413;N62522; AA627522; R11635; AA320408; AA379112; R09841; Z20320; N79421;D45800; T98959; AA217290; N30197; AA286132; and AA633983 (herebyincorporated by reference in their entirety.) However, to list everyrelated sequence would be cumbersome. Accordingly, preferably excludedfrom the present invention are one or more polynucleotides comprising,or alternatively consisting of, a nucleotide sequence described by thegeneral formula of a-b, where a is any integer between 1 to 3206 of SEQID NO:1, b is an integer of 15 to 3220, where both a and b correspond tothe positions of nucleotide residues shown in SEQ ID NO:1, and where theb is greater than or equal to a+14.

Epitope-Bearing Portions

The present invention encompasses polypeptides comprising, oralternatively consisting of, an epitope of the polypeptide having anamino acid sequence of SEQ ID NO:2, or an epitope of the polypeptidesequence encoded by a polynucleotide sequence contained in depositedclone HDPJO39 (ATCC Deposit No. 209623) or encoded by a polynucleotidethat hybridizes to the complement of the sequence of SEQ ID NO:1 orcontained in deposited clone HDPJO39 under stringent hybridizationconditions or lower stringency hybridization conditions as definedsupra. The present invention further encompasses polynucleotidesequences encoding an epitope of a polypeptide sequence of the invention(such as, for example, the sequence disclosed in SEQ ID NO:1),polynucleotide sequences of the complementary strand of a polynucleotidesequence encoding an epitope of the invention, and polynucleotidesequences which hybridize to the complementary strand under stringenthybridization conditions or lower stringency hybridization conditionsdefined supra.

The term “epitopes,” as used herein, refers to portions of a polypeptidehaving antigenic or immunogenic activity in an animal, preferably amammal, and most preferably in a human. In a preferred embodiment, thepresent invention encompasses a polypeptide comprising an epitope, aswell as the polynucleotide encoding this polypeptide. An “immunogenicepitope,” as used herein, is defined as a portion of a protein thatelicits an antibody response in an animal, as determined by any methodknown in the art, for example, by the methods for generating antibodiesdescribed infra. (See, for example, Geysen et al., Proc. Natl. Acad.Sci. USA 81:3998-4002 (1983)). The term “antigenic epitope,” as usedherein, is defined as a portion of a protein to which an antibody canimmunospecifically bind its antigen as determined by any method wellknown in the art, for example, by the immunoassays described herein.Immunospecific binding excludes non-specific binding but does notnecessarily exclude cross-reactivity with other antigens. Antigenicepitopes need not necessarily be immunogenic.

Fragments that function as epitopes may be produced by any conventionalmeans. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 82:5131-5135(1985), further described in U.S. Pat. No. 4,631,211).

In the present invention, antigenic epitopes preferably contain asequence of at least 4, at least 5, at least 6, at least 7, morepreferably at least 8, at least 9, at least 10, at least 15, at least20, at least 25, and, most preferably, between about 15 to about 30amino acids. Preferred polypeptides comprising immunogenic or antigenicepitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65,70, 75, 80, 85, 90, 95, or 100 amino acid residues in length. Antigenicepitopes are useful, for example, to raise antibodies, includingmonoclonal antibodies, that specifically bind the epitope. Antigenicepitopes can be used as the target molecules in immunoassays. (See, forinstance, Wilson et al., Cell 37:767-778 (1984); Sutcliffe et al.,Science 219:660-666 (1983)).

Similarly, immunogenic epitopes can be used, for example, to induceantibodies according to methods well known in the art. (See, forinstance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al.,Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol.66:2347-2354 (1985). In one embodiment, a preferred immunogenic epitopeincludes the secreted protein. The polypeptides comprising one or moreimmunogenic epitopes may be presented for eliciting an antibody responsetogether with a carrier protein, such as an albumin, to an animal system(such as, for example, rabbit or mouse), or, if the polypeptide is ofsufficient length (at least about 25 amino acids), the polypeptide maybe presented without a carrier. However, immunogenic epitopes comprisingas few as 8 to 10 amino acids have been shown to be sufficient to raiseantibodies capable of binding to, at the very least, linear epitopes ina denatured polypeptide (e.g., in Western blotting).

Epitope-bearing polypeptides of the present invention may be used toinduce antibodies according to methods well known in the art including,but not limited to, in vivo immunization, in vitro immunization, andphage display methods. See, e.g., Sutcliffe et al., supra; Wilson etal., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985). Ifin vivo immunization is used, animals may be immunized with freepeptide; however, anti-peptide antibody titer may be boosted by couplingthe peptide to a macromolecular carrier, such as keyhole limpethemacyanin (KLH) or tetanus toxoid. For instance, peptides containingcysteine residues may be coupled to a carrier using a linker such asmaleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptidesmay be coupled to carriers using a more general linking agent such asglutaraldehyde. Animals such as, for example, rabbits, rats, and miceare immunized with either free or carrier-coupled peptides, forinstance, by intraperitoneal and/or intradermal injection of emulsionscontaining about 100 micrograms of peptide or carrier protein andFreund's adjuvant or any other adjuvant known for stimulating an immuneresponse. Several booster injections may be needed, for instance, atintervals of about two weeks, to provide a useful titer of anti-peptideantibody that can be detected, for example, by ELISA assay using freepeptide adsorbed to a solid surface. The titer of anti-peptideantibodies in serum from an immunized animal may be increased byselection of anti-peptide antibodies, for instance, by adsorption to thepeptide on a solid support and elution of the selected antibodiesaccording to methods well known in the art.

Epitope bearing peptides of the invention may also be synthesized asmultiple antigen peptides (MAPs). MAPs consist of multiple copies of aspecific peptide attached to a non-immunogenic lysine core. By way ofnon-limiting example, MAPs may be synthesized onto a lysine core matrixattached to a polyethylene glycol-polystyrene (PEG-PS) support. Thepeptide of choice is synthesized onto the lysine residues using9-fluorenylmethoxycarbonyl (Fmoc) chemistry. For example, PerSeptiveBiosystems (Foster City, Calif.) offers MAPs supports such as the([Fmoc-Lys(Aloc)]₄-[Lys]₂-Lys-□Ala-PAl-PEG-PS) support which can be usedto synthesize MAPs. Cleavage of MAPs from the resin is performed withstandard trifloroacetic acid (TFA)-based cocktails. Purification ofMAPs, except for desalting, is not necessary.

As one of skill in the art will appreciate, and as discussed above, thepolypeptides of the present invention (e.g., those comprising animmunogenic or antigenic epitope) can be fused to heterologouspolypeptide sequences. For example, the polypeptides of the presentinvention (including fragments or variants thereof), may be fused withthe constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portionsthereof (CH1, CH2, CH3, or any combination thereof and portions thereof)resulting in chimeric polypeptides. By way of another non-limitingexample, polypeptides and/or antibodies of the present invention(including fragments or variants thereof) may be fused with albumin(including but not limited to recombinant human serum albumin orfragments or variants thereof (see, e.g., U.S. Pat. No. 5,876,969,issued Mar. 2, 1999, EP Patent 0 413 622, and U.S. Pat. No. 5,766,883,issued Jun. 16, 1998, herein incorporated by reference in theirentirety)). In a preferred embodiment, polypeptides and/or antibodies ofthe present invention (including fragments or variants thereof) arefused with the mature form of human serum albumin (i.e., amino acids1-585 of human serum albumin as shown in FIGS. 1 and 2 of EP Patent 0322 094) which is herein incorporated by reference in its entirety. Inanother preferred embodiment, polypeptides and/or antibodies of thepresent invention (including fragments or variants thereof) are fusedwith polypeptide fragments comprising, or alternatively consisting of,amino acid residues 1-z of human serum albumin, where z is an integerfrom 369 to 419, as described in U.S. Pat. No. 5,766,883 hereinincorporated by reference in its entirety. Polypeptides and/orantibodies of the present invention (including fragments or variantsthereof) may be fused to either the N- or C-terminal end of theheterologous protein (e.g., immunoglobulin Fc polypeptide or human serumalbumin polypeptide). Polynucleotides encoding fusion proteins of theinvention are also encompassed by the invention.

Such fusion proteins (as those described above) may facilitatepurification and may increase half-life in vivo. This has been shown forchimeric proteins consisting of the first two domains of the humanCD4-polypeptide and various domains of the constant regions of the heavyor light chains of mammalian immunoglobulins. See, e.g., EP 394,827;Traunecker et al., Nature, 331:84-86 (1988). Enhanced delivery of anantigen across the epithelial barrier to the immune system has beendemonstrated for antigens (e.g., insulin) conjugated to an FcRn bindingpartner such as IgG or Fc fragments (see, e.g., PCT Publications WO96/22024 and WO 99/04813). IgG Fusion proteins that have adisulfide-linked dimeric structure due to the IgG portion disulfidebonds have also been found to be more efficient in binding andneutralizing other molecules than monomeric polypeptides or fragmentsthereof alone. See, e.g., Fountoulakis et al., J. Biochem.,270:3958-3964 (1995). Nucleic acids encoding the above epitopes can alsobe recombined with a gene of interest as an epitope tag (e.g., thehemagglutinin (“HA”) tag or flag tag) to aid in detection andpurification of the expressed polypeptide. For example, a systemdescribed by Janknecht et al. allows for the ready purification ofnon-denatured fusion proteins expressed in human cell lines (Janknechtet al., 1991, Proc. Natl. Acad. Sci. USA 88:8972-897). In this system,the gene of interest is subcloned into a vaccinia recombination plasmidsuch that the open reading frame of the gene is translationally fused toan amino-terminal tag consisting of six histidine residues. The tagserves as a matrix-binding domain for the fusion protein. Extracts fromcells infected with the recombinant vaccinia virus are loaded onto Ni²⁺nitriloacetic acid-agarose column and histidine-tagged proteins can beselectively eluted with imidazole-containing buffers.

Additional fusion proteins of the invention may be generated through thetechniques of gene-shuffling, motif-shuffling, exon-shuffling, and/orcodon-shuffling (collectively referred to as “DNA shuffling”). DNAshuffling may be employed to modulate the activities of polypeptides ofthe invention, such methods can be used to generate polypeptides withaltered activity, as well as agonists and antagonists of thepolypeptides. See, generally, U.S. Pat. Nos. 5,605,793; 5,811,238;5,830,721; 5,834,252; and 5,837,458, and Patten et al., Curr. OpinionBiotechnol. 8:724-33 (1997); Harayama, Trends Biotechnol. 16(2):76-82(1998); Hansson, et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzoand Blasco, Biotechniques 24(2):308-13 (1998) (each of these patents andpublications are hereby incorporated by reference in its entirety). Inone embodiment, alteration of polynucleotides corresponding to SEQ IDNO:1 and the polypeptides encoded by these polynucleotides may beachieved by DNA shuffling. DNA shuffling involves the assembly of two ormore DNA segments by homologous or site-specific recombination togenerate variation in the polynucleotide sequence. In anotherembodiment, polynucleotides of the invention, or the encodedpolypeptides, may be altered by being subjected to random mutagenesis byerror-prone PCR, random nucleotide insertion or other methods prior torecombination. In another embodiment, one or more components, motifs,sections, parts, domains, fragments, etc., of a polynucleotide coding apolypeptide of the invention may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules.

Antibodies

Further polypeptides of the invention relate to antibodies and T-cellantigen receptors (TCR) which immunospecifically bind a polypeptide,polypeptide fragment, or variant of SEQ ID NO:2, and/or an epitope, ofthe present invention (as determined by immunoassays well known in theart for assaying specific antibody-antigen binding). Antibodies of theinvention include, but are not limited to, polyclonal, monoclonal,multispecific, human, humanized or chimeric antibodies, single chainantibodies, Fab fragments, F(ab′) fragments, fragments produced by a Fabexpression library, anti-idiotypic (anti-Id) antibodies (including,e.g., anti-id antibodies to antibodies of the invention), andepitope-binding fragments of any of the above. The term “antibody,” asused herein, refers to immunoglobulin molecules and immunologicallyactive portions of immunoglobulin molecules, i.e., molecules thatcontain an antigen binding site that immunospecifically binds anantigen. The immunoglobulin molecules of the invention can be of anytype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgG1, IgG2,IgG3, IgG4, IgA1 and IgA2) or subclass of immunoglobulin molecule.Immunoglobulins may have both a heavy and light chain. An array of IgG,IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a lightchain of the kappa or lambda forms. In a specific embodiment, theimmunoglobulin molecules of the invention are IgG1. In another specificembodiment, the immunoglobulin molecules of the invention are IgG4.

Most preferably the antibodies are human antigen-binding antibodyfragments of the present invention and include, but are not limited to,Fab, Fab′ and F(ab′)₂, Fd, single-chain Fvs (scFv), single-chainantibodies, disulfide-linked Fvs (sdFv) and fragments comprising eithera VL or VH domain. Antigen-binding antibody fragments, includingsingle-chain antibodies, may comprise the variable region(s) alone or incombination with the entirety or a portion of the following: hingeregion, CH1, CH2, and CH3 domains. Also included in the invention areantigen-binding fragments also comprising any combination of variableregion(s) with a hinge region, CH1, CH2, and CH3 domains. The antibodiesof the invention may be from any animal origin including birds andmammals. Preferably, the antibodies are human, murine (e.g., mouse andrat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.As used herein, “human” antibodies include antibodies having the aminoacid sequence of a human immunoglobulin and include antibodies isolatedfrom human immunoglobulin libraries or from animals transgenic for oneor more human immunoglobulin and that do not express endogenousimmunoglobulins, as described infra and, for example in, U.S. Pat. No.5,939,598 by Kucherlapati et al.

The antibodies of the present invention may be monospecific, bispecific,trispecific or of greater multispecificity. Multispecific antibodies maybe specific for different epitopes of a polypeptide of the presentinvention or may be specific for both a polypeptide of the presentinvention as well as for a heterologous epitope, such as a heterologouspolypeptide or solid support material. See, e.g., PCT publications WO93/17715; WO 92/08802; WO91/00360; WO 92/05793; Tutt, et al., J.Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893; 4,714,681;4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

Antibodies of the present invention may be described or specified interms of the epitope(s) or portion(s) of a polypeptide of the presentinvention which they recognize or specifically bind. The epitope(s) orpolypeptide portion(s) may be specified as described herein, e.g., byN-terminal and C-terminal positions, by size in contiguous amino acidresidues, or listed in the Tables and Figures. Antibodies whichspecifically bind any epitope or polypeptide of the present inventionmay also be excluded. Therefore, the present invention includesantibodies that specifically bind polypeptides of the present invention,and allows for the exclusion of the same.

In preferred, nonexclusive embodiments, the antibodies of the inventioninhibit one or more biological activities of D-SLAM polypeptides of theinvention through specific binding. In more preferred embodiments, theantibody of the invention inhibits D-SLAM-mediated inhibition of B cellproliferation. In additional preferred embodiments, the antibody of theinvention stimulates B cell proliferation. In additional preferredembodiments, the antibody of the invention is used in combination withone or more additional antibodies and/or polypeptides and/or otheragents to regulate B cell proliferation and/or biological phenomenarelated thereto.

Antibodies of the present invention may also be described or specifiedin terms of their cross-reactivity. Antibodies that do not bind anyother analog, ortholog, or homolog of a polypeptide of the presentinvention are included. Antibodies that bind polypeptides with at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 65%, at least 60%, at least 55%, and at least 50% identity(as calculated using methods known in the art and described herein) to apolypeptide of the present invention are also included in the presentinvention. In specific embodiments, antibodies of the present inventioncross-react with murine, rat and/or rabbit homologs of human proteinsand the corresponding epitopes thereof. Antibodies that do not bindpolypeptides with less than 95%, less than 90%, less than 85%, less than80%, less than 75%, less than 70%, less than 65%, less than 60%, lessthan 55%, and less than 50% identity (as calculated using methods knownin the art and described herein) to a polypeptide of the presentinvention are also included in the present invention. In a specificembodiment, the above-described cross-reactivity is with respect to anysingle specific antigenic or immunogenic polypeptide, or combination(s)of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenicpolypeptides disclosed herein. Further included in the present inventionare antibodies which bind polypeptides encoded by polynucleotides whichhybridize to a polynucleotide of the present invention underhybridization conditions (as described herein). Antibodies of thepresent invention may also be described or specified in terms of theirbinding affinity to a polypeptide of the invention. Preferred bindingaffinities include those with a dissociation constant or Kd less than5×10⁻² M, 10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁻⁴ M, 10⁻⁴ M, 5×10⁻⁵ M, 10⁻⁵ M,5×10⁻⁶ M, 10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M,5×10⁻¹⁰ M, 10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M,10⁻¹³ M, 5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, or 10⁻¹⁵ M.

The invention also provides antibodies that competitively inhibitbinding of an antibody to an epitope of the invention as determined byany method known in the art for determining competitive binding, forexample, the immunoassays described herein. In preferred embodiments,the antibody competitively inhibits binding to the epitope by at least95%, at least 90%, at least 85%, at least 80%, at least 75%, at least70%, at least 60%, or at least 50%.

Antibodies of the present invention may act as agonists or antagonistsof the polypeptides of the present invention. For example, the presentinvention includes antibodies which disrupt the receptor/ligandinteractions with the polypeptides of the invention either partially orfully. Preferably, antibodies of the present invention bind an antigenicepitope disclosed herein, or a portion thereof. The invention featuresboth receptor-specific antibodies and ligand-specific antibodies. Theinvention also features receptor-specific antibodies which do notprevent ligand binding but prevent receptor activation. Receptoractivation (i.e., signaling) may be determined by techniques describedherein or otherwise known in the art. For example, receptor activationcan be determined by detecting the phosphorylation (e.g., tyrosine orserine/threonine) of the receptor or its substrate byimmunoprecipitation followed by western blot analysis (for example, asdescribed supra). In specific embodiments, antibodies are provided thatinhibit ligand activity or receptor activity by at least 95%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least60%, or at least 50% of the activity in absence of the antibody.

The invention also features receptor-specific antibodies which bothprevent ligand binding and receptor activation as well as antibodiesthat recognize the receptor-ligand complex, and, preferably, do notspecifically recognize the unbound receptor or the unbound ligand.Likewise, included in the invention are neutralizing antibodies whichbind the ligand and prevent binding of the ligand to the receptor, aswell as antibodies which bind the ligand, thereby preventing receptoractivation, but do not prevent the ligand from binding the receptor.Further included in the invention are antibodies which activate thereceptor. These antibodies may act as receptor agonists, i.e.,potentiate or activate either all or a subset of the biologicalactivities of the ligand-mediated receptor activation, for example, byinducing dimerization of the receptor. The antibodies may be specifiedas agonists, antagonists or inverse agonists for biological activitiescomprising the specific biological activities of the peptides of theinvention disclosed herein. The above antibody agonists can be madeusing methods known in the art. See, e.g., PCT publication WO 96/40281;U.S. Pat. No. 5,811,097; Deng et al., Blood 92(6):1981-1988 (1998); Chenet al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol.161(4):1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214(1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al.,J. Cell. Sci. 111(Pt2):237-247 (1998); Pitard et al., J. Immunol.Methods 205(2):177-190 (1997); Liautard et al., Cytokine 9(4):233-241(1997); Carlson et al., J. Biol. Chem. 272(17):11295-11301 (1997);Taryman et al., Neuron 14(4):755-762 (1995); Muller et al., Structure6(9):1153-1167 (1998); Bartunek et al., Cytokine 8(1):14-20 (1996)(which are all incorporated by reference herein in their entireties).

Antibodies of the present invention may be used, for example, but notlimited to, to purify, detect, and target the polypeptides of thepresent invention, including both in vitro and in vivo diagnostic andtherapeutic methods. For example, the antibodies have use inimmunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides of the present invention in biological samples. See,e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold SpringHarbor Laboratory Press, 2nd ed. 1988) (incorporated by reference hereinin its entirety).

As discussed in more detail below, the antibodies of the presentinvention may be used either alone or in combination with othercompositions. The antibodies may further be recombinantly fused to aheterologous polypeptide at the N- or C-terminus or chemicallyconjugated (including covalently and non-covalently conjugations) topolypeptides or other compositions. For example, antibodies of thepresent invention may be recombinantly fused or conjugated to moleculesuseful as labels in detection assays and effector molecules such asheterologous polypeptides, drugs, radionuclides, or toxins. See, e.g.,PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Pat. No.5,314,995; and EP 396,387.

The antibodies of the invention include derivatives that are modified,i.e, by the covalent attachment of any type of molecule to the antibodysuch that covalent attachment does not prevent the antibody fromgenerating an anti-idiotypic response. For example, but not by way oflimitation, the antibody derivatives include antibodies that have beenmodified, e.g., by glycosylation, acetylation, pegylation,phosphorylation, amidation, derivatization by known protecting/blockinggroups, proteolytic cleavage, linkage to a cellular ligand or otherprotein, etc. Any of numerous chemical modifications may be carried outby known techniques, including, but not limited to specific chemicalcleavage, acetylation, formylation, metabolic synthesis of tunicamycin,etc. Additionally, the derivative may contain one or more non-classicalamino acids.

The antibodies of the present invention may be generated by any suitablemethod known in the art. Polyclonal antibodies to an antigen-of-interestcan be produced by various procedures well known in the art. Forexample, a polypeptide of the invention can be administered to varioushost animals including, but not limited to, rabbits, mice, rats, etc. toinduce the production of sera containing polyclonal antibodies specificfor the antigen. Various adjuvants may be used to increase theimmunological response, depending on the host species, and include butare not limited to, Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,keyhole limpet hemocyanins, dinitrophenol, and potentially useful humanadjuvants such as BCG (bacille Calmette-Guerin) and corynebacteriumparvum. Such adjuvants are also well known in the art.

Monoclonal antibodies can be prepared using a wide variety of techniquesknown in the art including the use of hybridoma, recombinant, and phagedisplay technologies, or a combination thereof. For example, monoclonalantibodies can be produced using hybridoma techniques including thoseknown in the art and taught, for example, in Harlow et al., Antibodies:A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed.1988); Hammerling, et al., in: Monoclonal Antibodies and T-CellHybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporatedby reference in their entireties). The term “monoclonal antibody” asused herein is not limited to antibodies produced through hybridomatechnology. The term “monoclonal antibody” refers to an antibody that isderived from a single clone, including any eukaryotic, prokaryotic, orphage clone, and not the method by which it is produced.

A “monoclonal antibody” may comprise, or alternatively consist of, twoproteins, i.e., a heavy and a light chain.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art and arediscussed in detail in the Examples (e.g., Example 11). In anon-limiting example, mice can be immunized with a polypeptide of theinvention or a cell expressing such peptide. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well-known techniques to any suitablemyeloma cells, for example cells from cell line SP20 available from theATCC. Hybridomas are selected and cloned by limited dilution. Thehybridoma clones are then assayed by methods known in the art for cellsthat secrete antibodies capable of binding a polypeptide of theinvention. Ascites fluid, which generally contains high levels ofantibodies, can be generated by immunizing mice with positive hybridomaclones.

Accordingly, the present invention provides methods of generatingmonoclonal antibodies as well as antibodies produced by the methodcomprising culturing a hybridoma cell secreting an antibody of theinvention wherein, preferably, the hybridoma is generated by fusingsplenocytes isolated from a mouse immunized with an antigen of theinvention with myeloma cells and then screening the hybridomas resultingfrom the fusion for hybridoma clones that secrete an antibody able tobind a polypeptide of the invention.

Antibody fragments which recognize specific epitopes may be generated byknown techniques. For example, Fab and F(ab′)₂ fragments of theinvention may be produced by proteolytic cleavage of immunoglobulinmolecules, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)₂ fragments). F(ab′)₂ fragments contain thevariable region, the light chain constant region and the CH1 domain ofthe heavy chain.

For example, the antibodies of the present invention can also begenerated using various phage display methods known in the art. In phagedisplay methods, functional antibody domains are displayed on thesurface of phage particles which carry the polynucleotide sequencesencoding them. In a particular embodiment, such phage can be utilized todisplay antigen-binding domains expressed from a repertoire orcombinatorial antibody library (e.g., human or murine). Phage expressingan antigen binding domain that binds the antigen of interest can beselected or identified with antigen, e.g., using labeled antigen orantigen bound or captured to a solid surface or bead. Phage used inthese methods are typically filamentous phage including fd and M13binding domains expressed from phage with Fab, Fv or disulfidestabilized Fv antibody domains recombinantly fused to either the phagegene III or gene VIII protein. Examples of phage display methods thatcan be used to make the antibodies of the present invention includethose disclosed in Brinkman et al., J. Immunol. Methods 182:41-50(1995); Ames et al., J. Immunol. Methods 184:177-186 (1995);Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persic et al.,Gene 187 9-18 (1997); Burton et al., Advances in Immunology 57:191-280(1994); PCT application No. PCT/GB91/01134; PCT publications WO90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired antigen binding fragment, and expressed in any desired host,including mammalian cells, insect cells, plant cells, yeast, andbacteria, e.g., as described in detail below. For example, techniques torecombinantly produce Fab, Fab′ and F(ab′)₂ fragments can also beemployed using methods known in the art such as those disclosed in PCTpublication WO 92/22324; Mullinax et al., BioTechniques 12(6):864-869(1992); and Sawai et al., AJRI 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988) (said references incorporated by referencein their entireties).

Examples of techniques which can be used to produce single-chain Fvs andantibodies include those described in U.S. Pat. Nos. 4,946,778 and5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu etal., PNAS 90:7995-7999 (1993); and Skerra et al., Science 240:1038-1040(1988). For some uses, including in vivo use of antibodies in humans andin vitro detection assays, it may be preferable to use chimeric,humanized, or human antibodies. A chimeric antibody is a molecule inwhich different portions of the antibody are derived from differentanimal species, such as antibodies having a variable region derived froma murine monoclonal antibody and a human immunoglobulin constant region.Methods for producing chimeric antibodies are known in the art. Seee.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214(1986); Gillies et al., (1989) J. Immunol. Methods 125:191-202; U.S.Pat. Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporatedherein by reference in their entirety. Humanized antibodies are antibodymolecules from non-human species antibody that binds the desired antigenhaving one or more complementarity determining regions (CDRs) from thenon-human species and a framework region from a human immunoglobulinmolecule. Often, framework residues in the human framework regions willbe substituted with the corresponding residue from the CDR donorantibody to alter, preferably improve, antigen binding. These frameworksubstitutions are identified by methods well known in the art, e.g., bymodeling of the interactions of the CDR and framework residues toidentify framework residues important for antigen binding and sequencecomparison to identify unusual framework residues at particularpositions. (See, e.g., Queen et al., U.S. Pat. No. 5,585,089; Riechmannet al., Nature 332:323 (1988), which are incorporated herein byreference in their entireties.) Antibodies can be humanized using avariety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101; and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Molecular Immunology 28(4/5):489-498(1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994);Roguska. et al., PNAS 91:969-973 (1994)), and chain shuffling (U.S. Pat.No. 5,565,332).

Completely human antibodies are particularly desirable for therapeutictreatment of human patients. Human antibodies can be made by a varietyof methods known in the art including phage display methods describedabove using antibody libraries derived from human immunoglobulinsequences. See also, U.S. Pat. Nos. 4,444,887 and 4,716,111; and PCTpublications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO96/34096, WO 96/33735, and WO 91/10741; each of which is incorporatedherein by reference in its entirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For example, the human heavy andlight chain immunoglobulin gene complexes may be introduced randomly orby homologous recombination into mouse embryonic stem cells.Alternatively, the human variable region, constant region, and diversityregion may be introduced into mouse embryonic stem cells in addition tothe human heavy and light chain genes. The mouse heavy and light chainimmunoglobulin genes may be rendered non-functional separately orsimultaneously with the introduction of human immunoglobulin loci byhomologous recombination. In particular, homozygous deletion of the JHregion prevents endogenous antibody production. The modified embryonicstem cells are expanded and microinjected into blastocysts to producechimeric mice. The chimeric mice are then bred to produce homozygousoffspring which express human antibodies. The transgenic mice areimmunized in the normal fashion with a selected antigen, e.g., all or aportion of a polypeptide of the invention. Monoclonal antibodiesdirected against the antigen can be obtained from the immunized,transgenic mice using conventional hybridoma technology. The humanimmunoglobulin transgenes harbored by the transgenic mice rearrangeduring B cell differentiation, and subsequently undergo class switchingand somatic mutation. Thus, using such a technique, it is possible toproduce therapeutically useful IgG, IgA, IgM and IgE antibodies. For anoverview of this technology for producing human antibodies, see Lonbergand Huszar, Int. Rev. Immunol. 13:65-93 (1995). For a detaileddiscussion of this technology for producing human antibodies and humanmonoclonal antibodies and protocols for producing such antibodies, see,e.g., PCT publications WO 98/24893; WO 92/01047; WO 96/34096; WO96/33735; European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923;5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318;5,885,793; 5,916,771; and 5,939,598, which are incorporated by referenceherein in their entirety. In addition, companies such as Abgenix, Inc.(Freemont, Calif.) and Genpharm (San Jose, Calif.) can be engaged toprovide human antibodies directed against a selected antigen usingtechnology similar to that described above.

Completely human antibodies which recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope. (Jespers et al., Bio/technology 12:899-903(1988)).

Further, antibodies to the polypeptides of the invention can, in turn,be utilized to generate anti-idiotype antibodies that “mimic”polypeptides of the invention using techniques well known to thoseskilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444;(1989) and Nissinoff, J. Immunol. 147(8):2429-2438 (1991)). For example,antibodies which bind to and competitively inhibit polypeptidemultimerization and/or binding of a polypeptide of the invention to aligand can be used to generate anti-idiotypes that “mimic” thepolypeptide multimerization and/or binding domain and, as a consequence,bind to and neutralize polypeptide and/or its ligand. Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize polypeptide ligand. For example, suchanti-idiotypic antibodies can be used to bind a polypeptide of theinvention and/or to bind its ligands/receptors, and thereby block itsbiological activity.

Polynucleotides Encoding Antibodies

The invention further provides polynucleotides comprising a nucleotidesequence encoding an antibody of the invention and fragments thereof.The invention also encompasses polynucleotides that hybridize understringent or lower stringency hybridization conditions, e.g., as definedsupra, to polynucleotides that encode an antibody, preferably, thatspecifically binds to a polypeptide of the invention, preferably, anantibody that binds to a polypeptide having the amino acid sequence ofSEQ ID NO:2.

The polynucleotides may be obtained, and the nucleotide sequence of thepolynucleotides determined, by any method known in the art. For example,if the nucleotide sequence of the antibody is known, a polynucleotideencoding the antibody may be assembled from chemically synthesizedoligonucleotides (e.g., as described in Kutmeier et al., BioTechniques17:242 (1994)), which, briefly, involves the synthesis of overlappingoligonucleotides containing portions of the sequence encoding theantibody, annealing and ligating of those oligonucleotides, and thenamplification of the ligated oligonucleotides by PCR.

Alternatively, a polynucleotide encoding an antibody may be generatedfrom nucleic acid from a suitable source. If a clone containing anucleic acid encoding a particular antibody is not available, but thesequence of the antibody molecule is known, a nucleic acid encoding theimmunoglobulin may be chemically synthesized or obtained from a suitablesource (e.g., an antibody cDNA library, or a cDNA library generatedfrom, or nucleic acid, preferably poly A+ RNA, isolated from, any tissueor cells expressing the antibody, such as hybridoma cells selected toexpress an antibody of the invention) by PCR amplification usingsynthetic primers hybridizable to the 3′ and 5′ ends of the sequence orby cloning using an oligonucleotide probe specific for the particulargene sequence to identify, e.g., a cDNA clone from a cDNA library thatencodes the antibody. Amplified nucleic acids generated by PCR may thenbe cloned into replicable cloning vectors using any method well known inthe art.

Once the nucleotide sequence and corresponding amino acid sequence ofthe antibody is determined, the nucleotide sequence of the antibody maybe manipulated using methods well known in the art for the manipulationof nucleotide sequences, e.g., recombinant DNA techniques, site directedmutagenesis, PCR, etc. (see, for example, the techniques described inSambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed.,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. and Ausubel etal., eds., 1998, Current Protocols in Molecular Biology, John Wiley &Sons, NY, which are both incorporated by reference herein in theirentireties), to generate antibodies having a different amino acidsequence, for example to create amino acid substitutions, deletions,and/or insertions.

In a specific embodiment, the amino acid sequence of the heavy and/orlight chain variable domains may be inspected to identify the sequencesof the complementarity determining regions (CDRs) by methods that arewell known in the art, e.g., by comparison to known amino acid sequencesof other heavy and light chain variable regions to determine the regionsof sequence hypervariability. Using routine recombinant DNA techniques,one or more of the CDRs may be inserted within framework regions, e.g.,into human framework regions to humanize a non-human antibody, asdescribed supra. The framework regions may be naturally occurring orconsensus framework regions, and preferably human framework regions(see, e.g., Chothia et al., J. Mol. Biol. 278: 457-479 (1998) for alisting of human framework regions). Preferably, the polynucleotidegenerated by the combination of the framework regions and CDRs encodesan antibody that specifically binds a polypeptide of the invention.Preferably, as discussed supra, one or more amino acid substitutions maybe made within the framework regions, and, preferably, the amino acidsubstitutions improve binding of the antibody to its antigen.Additionally, such methods may be used to make amino acid substitutionsor deletions of one or more variable region cysteine residuesparticipating in an intrachain disulfide bond to generate antibodymolecules lacking one or more intrachain disulfide bonds. Otheralterations to the polynucleotide are encompassed by the presentinvention and within the skill of the art.

In addition, techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. 81:851-855 (1984);Neuberger et al., Nature 312:604-608 (1984); Takeda et al., Nature314:452-454 (1985)) by splicing genes from a mouse antibody molecule ofappropriate antigen specificity together with genes from a humanantibody molecule of appropriate biological activity can be used. Asdescribed supra, a chimeric antibody is a molecule in which differentportions are derived from different animal species, such as those havinga variable region derived from a murine mAb and a human immunoglobulinconstant region, e.g., humanized antibodies.

Alternatively, techniques described for the production of single chainantibodies (U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988);Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); and Wardet al., Nature 334:544-54 (1989)) can be adapted to produce single chainantibodies. Single chain antibodies are formed by linking the heavy andlight chain fragments of the Fv region via an amino acid bridge,resulting in a single chain polypeptide. Techniques for the assembly offunctional Fv fragments in E. coli may also be used (Skerra et al.,Science 242:1038-1041 (1988)).

Methods of Producing Antibodies

The antibodies of the invention can be produced by any method known inthe art for the synthesis of antibodies, in particular, by chemicalsynthesis or preferably, by recombinant expression techniques.

Recombinant expression of an antibody of the invention, or fragment,derivative or analog thereof, (e.g., a heavy or light chain of anantibody of the invention or a single chain antibody of the invention),requires construction of an expression vector containing apolynucleotide that encodes the antibody. Once a polynucleotide encodingan antibody molecule or a heavy or light chain of an antibody, orportion thereof (preferably containing the heavy or light chain variabledomain), of the invention has been obtained, the vector for theproduction of the antibody molecule may be produced by recombinant DNAtechnology using techniques well known in the art. Thus, methods forpreparing a protein by expressing a polynucleotide containing anantibody encoding nucleotide sequence are described herein. Methodswhich are well known to those skilled in the art can be used toconstruct expression vectors containing antibody coding sequences andappropriate transcriptional and translational control signals. Thesemethods include, for example, in vitro recombinant DNA techniques,synthetic techniques, and in vivo genetic recombination. The invention,thus, provides replicable vectors comprising a nucleotide sequenceencoding an antibody molecule of the invention, or a heavy or lightchain thereof, or a heavy or light chain variable domain, operablylinked to a promoter. Such vectors may include the nucleotide sequenceencoding the constant region of the antibody molecule (see, e.g., PCTPublication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No.5,122,464) and the variable domain of the antibody may be cloned intosuch a vector for expression of the entire heavy or light chain.

The expression vector is transferred to a host cell by conventionaltechniques and the transfected cells are then cultured by conventionaltechniques to produce an antibody of the invention. Thus, the inventionincludes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, or a single chainantibody of the invention, operably linked to a heterologous promoter.In preferred embodiments for the expression of double-chainedantibodies, vectors encoding both the heavy and light chains may beco-expressed in the host cell for expression of the entireimmunoglobulin molecule, as detailed below.

A variety of host-expression vector systems may be utilized to expressthe antibody molecules of the invention. Such host-expression systemsrepresent vehicles by which the coding sequences of interest may beproduced and subsequently purified, but also represent cells which may,when transformed or transfected with the appropriate nucleotide codingsequences, express an antibody molecule of the invention in situ. Theseinclude but are not limited to microorganisms such as bacteria (e.g., E.coli, B. subtilis) transformed with recombinant bacteriophage DNA,plasmid DNA or cosmid DNA expression vectors containing antibody codingsequences; yeast (e.g., Saccharomyces, Pichia) transformed withrecombinant yeast expression vectors containing antibody codingsequences; insect cell systems infected with recombinant virusexpression vectors (e.g., baculovirus) containing antibody codingsequences; plant cell systems infected with recombinant virus expressionvectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus,TMV) or transformed with recombinant plasmid expression vectors (e.g.,Ti plasmid) containing antibody coding sequences; or mammalian cellsystems (e.g., COS, CHO, BHK, 293, 3T3 cells) harboring recombinantexpression constructs containing promoters derived from the genome ofmammalian cells (e.g., metallothionein promoter) or from mammalianviruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5Kpromoter). Preferably, bacterial cells such as Escherichia coli, andmore preferably, eukaryotic cells, especially for the expression ofwhole recombinant antibody molecule, are used for the expression of arecombinant antibody molecule. For example, mammalian cells such asChinese hamster ovary cells (CHO), in conjunction with a vector such asthe major intermediate early gene promoter element from humancytomegalovirus is an effective expression system for antibodies(Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2(1990)).

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the antibodymolecule being expressed. For example, when a large quantity of such aprotein is to be produced, for the generation of pharmaceuticalcompositions of an antibody molecule, vectors which direct theexpression of high levels of fusion protein products that are readilypurified may be desirable. Such vectors include, but are not limited, tothe E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791(1983)), in which the antibody coding sequence may be ligatedindividually into the vector in frame with the lac Z coding region sothat a fusion protein is produced; pIN vectors (Inouye & Inouye, NucleicAcids Res. 13:3101-3109 (1985); Van Heeke & Schuster, J. Biol. Chem.24:5503-5509 (1989)); and the like. pGEX vectors may also be used toexpress foreign polypeptides as fusion proteins with glutathioneS-transferase (GST). In general, such fusion proteins are soluble andcan easily be purified from lysed cells by adsorption and binding tomatrix glutathione-agarose beads followed by elution in the presence offree glutathione. The pGEX vectors are designed to include thrombin orfactor Xa protease cleavage sites so that the cloned target gene productcan be released from the GST moiety.

In an insect system, Autographa californica nuclear polyhedrosis virus(AcNPV) is used as a vector to express foreign genes. The virus grows inSpodoptera frugiperda cells. The antibody coding sequence may be clonedindividually into non-essential regions (for example the polyhedringene) of the virus and placed under control of an AcNPV promoter (forexample the polyhedrin promoter).

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, the antibody coding sequence of interest may be ligated to anadenovirus transcription/translation control complex, e.g., the latepromoter and tripartite leader sequence. This chimeric gene may then beinserted in the adenovirus genome by in vitro or in vivo recombination.Insertion in a non-essential region of the viral genome (e.g., region E1or E3) will result in a recombinant virus that is viable and capable ofexpressing the antibody molecule in infected hosts. (E.g., see Logan &Shenk, Proc. Natl. Acad. Sci. USA 81:355-359 (1984)). Specificinitiation signals may also be required for efficient translation ofinserted antibody coding sequences. These signals include the ATGinitiation codon and adjacent sequences. Furthermore, the initiationcodon must be in phase with the reading frame of the desired codingsequence to ensure translation of the entire insert. These exogenoustranslational control signals and initiation codons can be of a varietyof origins, both natural and synthetic. The efficiency of expression maybe enhanced by the inclusion of appropriate transcription enhancerelements, transcription terminators, etc. (see Bittner et al., Methodsin Enzymol. 153:51-544 (1987)).

In addition, a host cell strain may be chosen which modulates theexpression of the inserted sequences, or modifies and processes the geneproduct in the specific fashion desired. Such modifications (e.g.,glycosylation) and processing (e.g., cleavage) of protein products maybe important for the function of the protein. Different host cells havecharacteristic and specific mechanisms for the post-translationalprocessing and modification of proteins and gene products. Appropriatecell lines or host systems can be chosen to ensure the correctmodification and processing of the foreign protein expressed. To thisend, eukaryotic host cells which possess the cellular machinery forproper processing of the primary transcript, glycosylation, andphosphorylation of the gene product may be used. Such mammalian hostcells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK,293, 3T3, WI38, and in particular, breast cancer cell lines such as, forexample, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary glandcell line such as, for example, CRL7030 and Hs578Bst.

For long-term, high-yield production of recombinant proteins, stableexpression is preferred. For example, cell lines which stably expressthe antibody molecule may be engineered. Rather than using expressionvectors which contain viral origins of replication, host cells can betransformed with DNA controlled by appropriate expression controlelements (e.g., promoter, enhancer, sequences, transcriptionterminators, polyadenylation sites, etc.), and a selectable marker.Following the introduction of the foreign DNA, engineered cells may beallowed to grow for 1-2 days in an enriched media, and then are switchedto a selective media. The selectable marker in the recombinant plasmidconfers resistance to the selection and allows cells to stably integratethe plasmid into their chromosomes and grow to form foci which in turncan be cloned and expanded into cell lines. This method mayadvantageously be used to engineer cell lines which express the antibodymolecule. Such engineered cell lines may be particularly useful inscreening and evaluation of compounds that interact directly orindirectly with the antibody molecule.

A number of selection systems may be used, including but not limited tothe herpes simplex virus thymidine kinase (Wigler et al., Cell 11:223(1977)), hypoxanthine-guanine phosphoribosyltransferase (Szybalska &Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adeninephosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can beemployed in tk−, hgprt− or aprt− cells, respectively. Also,antimetabolite resistance can be used as the basis of selection for thefollowing genes: dhfr, which confers resistance to methotrexate (Wigleret al., Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl.Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance tomycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci. USA 78:2072(1981)); neo, which confers resistance to the aminoglycoside G-418Clinical Pharmacy 12:488-505; Wu and Wu, Biotherapy 3:87-95 (1991);Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993); Mulligan,Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev. Biochem.62:191-217 (1993); May, 1993, TIB TECH 11(5):155-215); and hygro, whichconfers resistance to hygromycin (Santerre et al., Gene 30:147 (1984)).Methods commonly known in the art of recombinant DNA technology may beroutinely applied to select the desired recombinant clone, and suchmethods are described, for example, in Ausubel et al. (eds.), CurrentProtocols in Molecular Biology, John Wiley & Sons, NY (1993); Kriegler,Gene Transfer and Expression, A Laboratory Manual, Stockton Press, NY(1990); and in Chapters 12 and 13, Dracopoli et al. (eds), CurrentProtocols in Human Genetics, John Wiley & Sons, NY (1994);Colberre-Garapin et al., J. Mol. Biol. 150:1 (1981), which areincorporated by reference herein in their entireties.

The expression levels of an antibody molecule can be increased by vectoramplification (for a review, see Bebbington and Hentschel, The use ofvectors based on gene amplification for the expression of cloned genesin mammalian cells in DNA cloning, Vol.3. (Academic Press, New York,1987)). When a marker in the vector system expressing antibody isamplifiable, increase in the level of inhibitor present in culture ofhost cell will increase the number of copies of the marker gene. Sincethe amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al., Mol. Cell. Biol. 3:257(1983)).

The host cell may be co-transfected with two expression vectors of theinvention, the first vector encoding a heavy chain derived polypeptideand the second vector encoding a light chain derived polypeptide. Thetwo vectors may contain identical selectable markers which enable equalexpression of heavy and light chain polypeptides. Alternatively, asingle vector may be used which encodes, and is capable of expressing,both heavy and light chain polypeptides. In such situations, the lightchain should be placed before the heavy chain to avoid an excess oftoxic free heavy chain (Proudfoot, Nature 322:52 (1986); Kohler, Proc.Natl. Acad. Sci. USA 77:2197 (1980)). The coding sequences for the heavyand light chains may comprise cDNA or genomic DNA.

Once an antibody molecule of the invention has been produced by ananimal, chemically synthesized, or recombinantly expressed, it may bepurified by any method known in the art for purification of animmunoglobulin molecule, for example, by chromatography (e.g., ionexchange, affinity, particularly by affinity for the specific antigenafter Protein A, and sizing column chromatography), centrifugation,differential solubility, or by any other standard technique for thepurification of proteins. In addition, the antibodies of the presentinvention or fragments thereof can be fused to heterologous polypeptidesequences described herein or otherwise known in the art, to facilitatepurification.

The present invention encompasses antibodies recombinantly fused orchemically conjugated (including both covalent and non-covalentconjugations) to a polypeptide (or portion thereof, preferably at least10, 20, 30, 40, 50, 60, 70, 80, 90 or 100 amino acids of thepolypeptide) of the present invention to generate fusion proteins. Thefusion does not necessarily need to be direct, but may occur throughlinker sequences. The antibodies may be specific for antigens other thanpolypeptides (or portion thereof, preferably at least 10, 20, 30, 40,50, 60, 70, 80, 90 or 100 amino acids of the polypeptide) of the presentinvention. For example, antibodies may be used to target thepolypeptides of the present invention to particular cell types, eitherin vitro or in vivo, by fusing or conjugating the polypeptides of thepresent invention to antibodies specific for particular cell surfacereceptors. Antibodies fused or conjugated to the polypeptides of thepresent invention may also be used in in vitro immunoassays andpurification methods using methods known in the art. See e.g., Harbor etal., supra, and PCT publication WO 93/21232; EP 439,095; Naramura etal., Immunol. Lett. 39:91-99 (1994); U.S. Pat. No. 5,474,981; Gillies etal., PNAS 89:1428-1432 (1992); Fell et al., J. Immunol.146:2446-2452(1991), which are incorporated by reference in theirentireties.

The present invention further includes compositions comprising thepolypeptides of the present invention fused or conjugated to antibodydomains other than the variable regions. For example, the polypeptidesof the present invention may be fused or conjugated to an antibody Fcregion, or portion thereof. The antibody portion fused to a polypeptideof the present invention may comprise the constant region, hinge region,CH1 domain, CH2 domain, and CH3 domain or any combination of wholedomains or portions thereof. The polypeptides may also be fused orconjugated to the above antibody portions to form multimers. Forexample, Fc portions fused to the polypeptides of the present inventioncan form dimers through disulfide bonding between the Fc portions.Higher multimeric forms can be made by fusing the polypeptides toportions of IgA and IgM. Methods for fusing or conjugating thepolypeptides of the present invention to antibody portions are known inthe art. See, e.g., U.S. Pat. Nos. 5,336,603; 5,622,929; 5,359,046;5,349,053; 5,447,851; 5,112,946; EP 307,434; EP 367,166; PCTpublications WO 96/04388; WO 91/06570; Ashkenazi et al., Proc. Natl.Acad. Sci. USA 88:10535-10539 (1991); Zheng et al., J. Immunol.154:5590-5600 (1995); and Vil et al., Proc. Natl. Acad. Sci. USA89:11337-11341(1992) (said references incorporated by reference in theirentireties).

As discussed, supra, the polypeptides corresponding to a polypeptide,polypeptide fragment, or a variant of SEQ ID NO:2 may be fused orconjugated to the above antibody portions to increase the in vivo halflife of the polypeptides or for use in immunoassays using methods knownin the art. Further, the polypeptides corresponding to SEQ ID NO:2 maybe fused or conjugated to the above antibody portions to facilitatepurification. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. (EP 394,827; Traunecker et al., Nature331:84-86 (1988). The polypeptides of the present invention fused orconjugated to an antibody having disulfide-linked dimeric structures(due to the IgG) may also be more efficient in binding and neutralizingother molecules, than the monomeric secreted protein or protein fragmentalone. (Fountoulakis et al., J. Biochem. 270:3958-3964 (1995)). In manycases, the Fc part in a fusion protein is beneficial in therapy anddiagnosis, and thus can result in, for example, improved pharmacokineticproperties. (EP A 232,262). Alternatively, deleting the Fc part afterthe fusion protein has been expressed, detected, and purified, would bedesired. For example, the Fc portion may hinder therapy and diagnosis ifthe fusion protein is used as an antigen for immunizations. In drugdiscovery, for example, human proteins, such as hIL-5, have been fusedwith Fc portions for the purpose of high-throughput screening assays toidentify antagonists of hIL-5. (See, Bennett et al., J. MolecularRecognition 8:52-58 (1995); Johanson et al., J. Biol. Chem.270:9459-9471 (1995).

Moreover, the antibodies or fragments thereof of the present inventioncan be fused to marker sequences, such as a peptide to facilitatepurification. In preferred embodiments, the marker amino acid sequenceis a hexa-histidine peptide, such as the tag provided in a pQE vector(QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), amongothers, many of which are commercially available. As described in Gentzet al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance,hexa-histidine provides for convenient purification of the fusionprotein. Other peptide tags useful for purification include, but are notlimited to, the “HA” tag, which corresponds to an epitope derived fromthe influenza hemagglutinin protein (Wilson et al., Cell 37:767 (1984))and the “flag” tag.

The present invention further encompasses antibodies or fragmentsthereof conjugated to a diagnostic or therapeutic agent. The antibodiescan be used diagnostically to, for example, monitor the development orprogression of a tumor as part of a clinical testing procedure to, e.g.,determine the efficacy of a given treatment regimen. Detection can befacilitated by coupling the antibody to a detectable substance. Examplesof detectable substances include various enzymes, prosthetic groups,fluorescent materials, luminescent materials, bioluminescent materials,radioactive materials, positron emitting metals using various positronemission tomographies, and nonradioactive paramagnetic metal ions. Thedetectable substance may be coupled or conjugated either directly to theantibody (or fragment thereof) or indirectly, through an intermediate(such as, for example, a linker known in the art) using techniques knownin the art. See, for example, U.S. Pat. No. 4,741,900 for metal ionswhich can be conjugated to antibodies for use as diagnostics accordingto the present invention. Examples of suitable enzymes includehorseradish peroxidase, alkaline phosphatase, beta-galactosidase, oracetylcholinesterase; examples of suitable prosthetic group complexesinclude streptavidin/biotin and avidin/biotin; examples of suitablefluorescent materials include umbelliferone, fluorescein, fluoresceinisothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansylchloride or phycoerythrin; an example of a luminescent material includesluminol; examples of bioluminescent materials include luciferase,luciferin, and aequorin; and examples of suitable radioactive materialinclude ¹²⁵I, ¹³¹I, ¹¹¹In or ⁹⁹Tc.

Further, an antibody or fragment thereof may be conjugated to atherapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidalagent, a therapeutic agent or a radioactive metal ion, e.g.,alpha-emitters such as, for example, ²¹³Bi. A cytotoxin or cytotoxicagent includes any agent that is detrimental to cells. Examples includepaclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine,mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin,doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone,mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids,procaine, tetracaine, lidocaine, propranolol, and puromycin and analogsor homologs thereof. Therapeutic agents include, but are not limited to,antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine,cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g.,mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) andlomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol,streptozotocin, mitomycin C, and cis-dichlorodiamine platinum (II) (DDP)cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

The conjugates of the invention can be used for modifying a givenbiological response, the therapeutic agent or drug moiety is not to beconstrued as limited to classical chemical therapeutic agents. Forexample, the drug moiety may be a protein or polypeptide possessing adesired biological activity. Such proteins may include, for example, atoxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin;a protein such as tumor necrosis factor, alpha-interferon,beta-interferon, nerve growth factor, platelet derived growth factor,tissue plasminogen activator, an apoptotic agent, e.g., TNF-alpha,TNF-beta, AIM I (See, International Publication No. WO 97/33899), AIM II(See, International Publication No. WO 97/34911), Fas Ligand (Takahashiet al., Int. Immunol., 6:1567-1574 (1994)), VEGI (See, InternationalPublication No. WO 99/23105), CD40 Ligand, a thrombotic agent or ananti-angiogenic agent, e.g., angiostatin, endostatin or VEGI (See,International Publication No. WO 99/23105); or, biological responsemodifiers such as, for example, lymphokines, interleukin-1 (“IL-1”),interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophagecolony stimulating factor (“GM-CSF”), granulocyte colony stimulatingfactor (“G-CSF”), or other growth factors.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or purification of the targetantigen. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene.

Techniques for conjugating such therapeutic moiety to antibodies arewell known, see, e.g., Arnon et al., “Monoclonal Antibodies ForImmunotargeting Of Drugs In Cancer Therapy”, in Monoclonal AntibodiesAnd Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss,Inc. 1985); Hellstrom et al., “Antibodies For Drug Delivery”, inControlled Drug Delivery (2nd Ed.), Robinson et al. (eds.), pp. 623-53(Marcel Dekker, Inc. 1987); Thorpe, “Antibody Carriers Of CytotoxicAgents In Cancer Therapy: A Review”, in Monoclonal Antibodies '84:Biological And Clinical Applications, Pinchera et al. (eds.), pp.475-506 (1985); “Analysis, Results, And Future Prospective Of TheTherapeutic Use Of Radiolabeled Antibody in Cancer Therapy”, inMonoclonal Antibodies For Cancer Detection And Therapy, Baldwin et al.(eds.), pp. 303-16 (Academic Press 1985), and Thorpe et al., “ThePreparation And Cytotoxic Properties Of Antibody-Toxin Conjugates”,Immunol. Rev. 62:119-58 (1982).

In specific embodiments, D-SLAM binding polypeptides of the inventionare attached either directly or indirectly, to macrocyclic chelatorsuseful for chelating radiometal ions, including but not limited to¹⁷⁷Lu, ⁹⁰Y, ¹⁶⁶Ho, and ¹⁵³Sm, to polypeptides. In a preferredembodiment, the radiometal ion associated with the macrocyclic chelatorsattached to D-SLAM polypeptides of the invention is ¹¹¹In. In anotherpreferred embodiment, the radiometal ion associated with the macrocyclicchelator attached to D-SLAM polypeptides of the invention is ⁹⁰Y. Inspecific embodiments, the macrocyclic chelator is1,4,7,10-tetraazacyclododecane-N,N′,N″,N′″-tetraacetic acid (DOTA). Inone embodiment the side chain moiety of one or more classical ornon-classical amino acids in a D-SLAM binding polypeptide comprises aDOTA molecule. In other specific embodiments, the DOTA is attached tothe D-SLAM binding polypeptide of the invention via a linker molecule.Examples of linker molecules useful for conjugating DOTA to apolypeptide are commonly known in the art—see, for example, DeNardo etal., Clin. Cancer Res., 4(10):2483-90 (1998); Peterson et al.,Bioconjug. Chem., 10(4):553-7 (1999); and Zimmerman et al, Nucl. Med.Biol., 26(8):943-50 (1999), which are hereby incorporated by referencein their entirety. In addition, U.S. Pat. Nos. 5,652,361 and 5,756,065,which disclose chelating agents that may be conjugated to antibodies,and methods for making and using them, are hereby incorporated byreference in their entireties. Though U.S. Pat. Nos. 5,652,361 and5,756,065 focus on conjugating chelating agents to antibodies, oneskilled in the art could readily adapt the methods disclosed therein inorder to conjugate chelating agents to other polypeptides.

Alternatively, an antibody can be conjugated to a second antibody toform an antibody heteroconjugate as described by Segal in U.S. Pat. No.4,676,980, which is incorporated herein by reference in its entirety.

An antibody, with or without a therapeutic moiety conjugated to it,administered alone or in combination with cytotoxic factor(s) and/orcytokine(s) can be used as a therapeutic.

Immunophenotyping

The antibodies of the invention may be utilized for immunophenotyping ofcell lines and biological samples. The translation product of the geneof the present invention may be useful as a cell specific marker, ormore specifically as a cellular marker that is differentially expressedat various stages of differentiation and/or maturation of particularcell types. Monoclonal antibodies directed against a specific epitope,or combination of epitopes, will allow for the screening of cellularpopulations expressing the marker. Various techniques can be utilizedusing monoclonal antibodies to screen for cellular populationsexpressing the marker(s), and include magnetic separation usingantibody-coated magnetic beads, “panning” with antibody attached to asolid matrix (i.e., plate), and flow cytometry (See, e.g., U.S. Pat. No.5,985,660; and Morrison et al., Cell, 96:737-49 (1999)).

These techniques allow for the screening of particular populations ofcells, such as might be found with hematological malignancies (i.e.minimal residual disease (MRD) in acute leukemic patients) and“non-self” cells in transplantations to prevent Graft-versus-HostDisease (GVHD). Alternatively, these techniques allow for the screeningof hematopoietic stem and progenitor cells capable of undergoingproliferation and/or differentiation, as might be found in humanumbilical cord blood.

Assays for Antibody Binding

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused, include but are not limited to, competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 1-4 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., western blot analysis. One of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or ¹²⁵I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected and to reduce the background noise. Forfurther discussion regarding western blot protocols see, e.g., Ausubelet al, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. One of skill inthe art would be knowledgeable as to the parameters that can be modifiedto increase the signal detected as well as other variations of ELISAsknown in the art. For further discussion regarding ELISAs see, e.g.,Ausubel et al, eds, 1994, Current Protocols in Molecular Biology, Vol.1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., ³H or ¹²⁵I)in the presence of increasing amounts of an unlabeled second antibody.

Therapeutic Uses

The present invention is further directed to antibody-based therapieswhich involve administering antibodies of the invention to an animal,preferably a mammal, and most preferably a human, patient for treatingone or more of the disclosed diseases, disorders, or conditions.Therapeutic compounds of the invention include, but are not limited to,antibodies of the invention (including fragments, analogs andderivatives thereof as described herein) and nucleic acids encodingantibodies of the invention (including fragments, analogs andderivatives thereof and anti-idiotypic antibodies as described herein).The antibodies of the invention can be used to treat, inhibit or preventdiseases, disorders or conditions associated with aberrant expressionand/or activity of a polypeptide of the invention, including, but notlimited to, any one or more of the diseases, disorders, or conditionsdescribed herein (e.g., autoimmune diseases, disorders, or conditionsassociated with such diseases or disorders, including, but not limitedto, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia,idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolyticanemia, antiphospholipid syndrome, dermatitis, allergicencephalomyelitis, myocarditis, relapsing polychondritis, rheumaticheart disease, glomerulonephritis (e.g, IgA nephropathy), MultipleSclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura(e.g., Henloch-Scoenlein purpura), Reiter's Disease, Stiff-Man Syndrome,Autoimmune Pulmonary Inflammation, Guillain-Barre Syndrome, insulindependent diabetes mellitis, and autoimmune inflammatory eye, autoimmunethyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis, systemiclupus erhythematosus, Goodpasture's syndrome, Pemphigus, Receptorautoimmunities such as, for example, (a) Graves' Disease, (b) MyastheniaGravis, and (c) insulin resistance, autoimmune hemolytic anemia,autoimmune thrombocytopenic purpura, rheumatoid arthritis, schlerodermawith anti-collagen antibodies, mixed connective tissue disease,polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison'sdisease, infertility, glomerulonephritis such as primaryglomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren'ssyndrome, diabetes millitus, and adrenergic drug resistance (includingadrenergic drug resistance with asthma or cystic fibrosis), chronicactive hepatitis, primary biliary cirrhosis, other endocrine glandfailure, vitiligo, vasculitis, post-MI, cardiotomy syndrome, urticaria,atopic dermatitis, asthma, inflammatory myopathies, and otherinflammatory, granulamatous, degenerative, and atrophic disorders).

In a specific embodiment, antibodies of the invention are be used totreat, inhibit, prognose, diagnose or prevent rheumatoid arthritis.

In another specific embodiment, antibodies of the invention are used totreat, inhibit, prognose, diagnose or prevent systemic lupuserythematosis.

The treatment and/or prevention of diseases, disorders, or conditionsassociated with aberrant expression and/or activity of a polypeptide ofthe invention includes, but is not limited to, alleviating symptomsassociated with those diseases, disorders or conditions. The antibodiesof the invention may also be used to target and kill cells expressingD-SLAM on their surface and/or cells having D-SLAM bound to theirsurface. Antibodies of the invention may be provided in pharmaceuticallyacceptable compositions as known in the art or as described herein.

A summary of the ways in which the antibodies of the present inventionmay be used therapeutically includes binding polynucleotides orpolypeptides of the present invention locally or systemically in thebody or by direct cytotoxicity of the antibody, e.g. as mediated bycomplement (CDC) or by effector cells (ADCC). Some of these approachesare described in more detail below. Armed with the teachings providedherein, one of ordinary skill in the art will know how to use theantibodies of the present invention for diagnostic, monitoring ortherapeutic purposes without undue experimentation.

The antibodies of this invention may be advantageously utilized incombination with other monoclonal or chimeric antibodies, or withlymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3and IL-7), for example, which serve to increase the number or activityof effector cells which interact with the antibodies.

The antibodies of the invention may be administered alone or incombination with other types of treatments (e.g., radiation therapy,chemotherapy, hormonal therapy, immunotherapy, anti-tumor agents,antibiotics, and immunoglobulin). Generally, administration of productsof a species origin or species reactivity (in the case of antibodies)that is the same species as that of the patient is preferred. Thus, in apreferred embodiment, human antibodies, fragments derivatives, analogs,or nucleic acids, are administered to a human patient for therapy orprophylaxis.

It is preferred to use high affinity and/or potent in vivo inhibitingand/or neutralizing antibodies against polypeptides or polynucleotidesof the present invention, fragments or regions thereof, for bothimmunoassays directed to and therapy of disorders related topolynucleotides or polypeptides, including fragments thereof, of thepresent invention. Such antibodies, fragments, or regions, willpreferably have an affinity for polynucleotides or polypeptides of theinvention, including fragments thereof. Preferred binding affinitiesinclude those with a dissociation constant or Kd less than 5×10⁻² M,10⁻² M, 5×10⁻³ M, 10⁻³ M, 5×10⁴ M, 10⁴ M, 5×10⁻⁵ M, 10⁻⁵ M, 5×10⁻⁶ M,10⁻⁶ M, 5×10⁻⁷ M, 10⁻⁷ M, 5×10⁻⁸ M, 10⁻⁸ M, 5×10⁻⁹ M, 10⁻⁹ M, 5×10⁻¹⁰ M,10⁻¹⁰ M, 5×10⁻¹¹ M, 10⁻¹¹ M, 5×10⁻¹² M, 10⁻¹² M, 5×10⁻¹³ M, 10⁻¹³ M,5×10⁻¹⁴ M, 10⁻¹⁴ M, 5×10⁻¹⁵ M, and 10¹⁵ M.

Gene Therapy

In a specific embodiment, nucleic acids comprising sequences encodingantibodies or functional derivatives thereof, are administered to treat,inhibit or prevent a disease or disorder associated with aberrantexpression and/or activity of a polypeptide of the invention, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration to a subject of an expressed or expressible nucleic acid.In this embodiment of the invention, the nucleic acids produce theirencoded protein that mediates a therapeutic effect.

Any of the methods for gene therapy available in the art can be usedaccording to the present invention. Exemplary methods are describedbelow.

For general reviews of the methods of gene therapy, see Goldspiel etal., Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, Ann. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, Ann. Rev.Biochem. 62:191-217 (1993); May, TIBTECH 11(5):155-215 (1993). Methodscommonly known in the art of recombinant DNA technology which can beused are described in Ausubel et al. (eds.), Current Protocols inMolecular Biology, John Wiley & Sons, NY (1993); and Kriegler, GeneTransfer and Expression, A Laboratory Manual, Stockton Press, NY (1990).

In a preferred embodiment, the compound comprises nucleic acid sequencesencoding an antibody, said nucleic acid sequences being part ofexpression vectors that express the antibody or fragments or chimericproteins or heavy or light chains thereof in a suitable host. Inparticular, such nucleic acid sequences have promoters operably linkedto the antibody coding region, said promoter being inducible orconstitutive, and, optionally, tissue-specific. In another particularembodiment, nucleic acid molecules are used in which the antibody codingsequences and any other desired sequences are flanked by regions thatpromote homologous recombination at a desired site in the genome, thusproviding for intrachromosomal expression of the antibody encodingnucleic acids (Koller and Smithies, Proc. Natl. Acad. Sci. USA86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989). Inspecific embodiments, the expressed antibody molecule is a single chainantibody; alternatively, the nucleic acid sequences include sequencesencoding both the heavy and light chains, or fragments thereof, of theantibody.

Delivery of the nucleic acids into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vectors, or indirect, in which case, cells arefirst transformed with the nucleic acids in vitro, then transplantedinto the patient. These two approaches are known, respectively, as invivo or ex vivo gene therapy.

In a specific embodiment, the nucleic acid sequences are directlyadministered in vivo, where it is expressed to produce the encodedproduct. This can be accomplished by any of numerous methods known inthe art, e.g., by constructing them as part of an appropriate nucleicacid expression vector and administering it so that they becomeintracellular, e.g., by infection using defective or attenuatedretrovirals or other viral vectors (see U.S. Pat. No. 4,980,286), or bydirect injection of naked DNA, or by use of microparticle bombardment(e.g., a gene gun; Biolistic, Dupont), or coating with lipids orcell-surface receptors or transfecting agents, encapsulation inliposomes, microparticles, or microcapsules, or by administering them inlinkage to a peptide which is known to enter the nucleus, byadministering it in linkage to a ligand subject to receptor-mediatedendocytosis (see, e.g., Wu and Wu, J. Biol. Chem. 262:4429-4432 (1987))(which can be used to target cell types specifically expressing thereceptors), etc. In another embodiment, nucleic acid-ligand complexescan be formed in which the ligand comprises a fusogenic viral peptide todisrupt endosomes, allowing the nucleic acid to avoid lysosomaldegradation. In yet another embodiment, the nucleic acid can be targetedin vivo for cell specific uptake and expression, by targeting a specificreceptor (see, e.g., PCT Publications WO 92/06180; WO 92/22635;WO92/20316; WO93/14188, WO 93/20221). Alternatively, the nucleic acidcan be introduced intracellularly and incorporated within host cell DNAfor expression, by homologous recombination (Koller and Smithies, Proc.Natl. Acad. Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature342:435-438 (1989)).

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an antibody of the invention are used. For example, aretroviral vector can be used (see Miller et al., Meth. Enzymol.217:581-599 (1993)). These retroviral vectors contain the componentsnecessary for the correct packaging of the viral genome and integrationinto the host cell DNA. The nucleic acid sequences encoding the antibodyto be used in gene therapy are cloned into one or more vectors, whichfacilitates delivery of the gene into a patient. More detail aboutretroviral vectors can be found in Boesen et al., Biotherapy 6:291-302(1994), which describes the use of a retroviral vector to deliver themdr1 gene to hematopoietic stem cells in order to make the stem cellsmore resistant to chemotherapy. Other references illustrating the use ofretroviral vectors in gene therapy are: Clowes et al., J. Clin. Invest.93:644-651 (1994); Kiem et al., Blood 83:1467-1473 (1994); Salmons andGunzberg, Human Gene Therapy 4:129-141 (1993); and Grossman and Wilson,Curr. Opin. in Genetics and Devel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). In apreferred embodiment, adenovirus vectors are used.

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993);U.S. Pat. No. 5,436,146).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see, e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Clin., Pharmac. Ther. 29:69-92m (1985) and may be used in accordancewith the present invention, provided that the necessary developmentaland physiological functions of the recipient cells are not disrupted.The technique should provide for the stable transfer of the nucleic acidto the cell, so that the nucleic acid is expressible by the cell andpreferably heritable and expressible by its cell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. Recombinant blood cells (e.g., hematopoieticstem or progenitor cells) are preferably administered intravenously. Theamount of cells envisioned for use depends on the desired effect,patient state, etc., and can be determined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include, but arenot limited to, epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient.

In an embodiment in which recombinant cells are used in gene therapy,nucleic acid sequences encoding an antibody are introduced into thecells such that they are expressible by the cells or their progeny, andthe recombinant cells are then administered in vivo for therapeuticeffect. In a specific embodiment, stem or progenitor cells are used. Anystem and/or progenitor cells which can be isolated and maintained invitro can potentially be used in accordance with this embodiment of thepresent invention (see e.g. PCT Publication WO 94/08598; Stemple andAnderson, Cell 71:973-985 (1992); Rheinwald, Meth. Cell Bio. 21A:229(1980); and Pittelkow and Scott, Mayo Clinic Proc. 61:771 (1986)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy comprises an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

Demonstration of Therapeutic or Prophylactic Activity

The compounds or pharmaceutical compositions of the invention arepreferably tested in vitro, and then in vivo for the desired therapeuticor prophylactic activity, prior to use in humans. For example, in vitroassays to demonstrate the therapeutic or prophylactic utility of acompound or pharmaceutical composition include, the effect of a compoundon a cell line or a patient tissue sample. The effect of the compound orcomposition on the cell line and/or tissue sample can be determinedutilizing techniques known to those of skill in the art including, butnot limited to, rosette formation assays and cell lysis assays. Inaccordance with the invention, in vitro assays which can be used todetermine whether administration of a specific compound is indicated,include in vitro cell culture assays in which a patient tissue sample isgrown in culture, and exposed to or otherwise administered a compound,and the effect of such compound upon the tissue sample is observed.

Therapeutic and/or Prophylactic Administration and Composition

The invention provides methods of treatment, inhibition and prophylaxisby administration to a subject of an effective amount of a compound orpharmaceutical composition of the invention, preferably an antibody ofthe invention. In a preferred embodiment, the compound is substantiallypurified (e.g., substantially free from substances that limit its effector produce undesired side effects). The subject is preferably an animal,including but not limited to animals such as cows, pigs, horses,chickens, cats, dogs, etc., and is preferably a mammal, and mostpreferably human.

Formulations and methods of administration that can be employed when thecompound comprises a nucleic acid or an immunoglobulin are describedabove; additional appropriate formulations and routes of administrationcan be selected from among those described herein below.

Various delivery systems are known and can be used to administer acompound of the invention, e.g., encapsulation in liposomes,microparticles, microcapsules, recombinant cells capable of expressingthe compound, receptor-mediated endocytosis (see, e.g., Wu and Wu, J.Biol. Chem. 262:4429-4432 (1987)), construction of a nucleic acid aspart of a retroviral or other vector, etc. Methods of introductioninclude but are not limited to intradermal, intramuscular,intraperitoneal, intravenous, subcutaneous, intranasal, epidural, andoral routes. The compounds or compositions may be administered by anyconvenient route, for example by infusion or bolus injection, byabsorption through epithelial or mucocutaneous linings (e.g., oralmucosa, rectal and intestinal mucosa, etc.) and may be administeredtogether with other biologically active agents. Administration can besystemic or local. In addition, it may be desirable to introduce thepharmaceutical compounds or compositions of the invention into thecentral nervous system by any suitable route, including intraventricularand intrathecal injection; intraventricular injection may be facilitatedby an intraventricular catheter, for example, attached to a reservoir,such as an Ommaya reservoir. Pulmonary administration can also beemployed, e.g., by use of an inhaler or nebulizer, and formulation withan aerosolizing agent.

In a specific embodiment, it may be desirable to administer thepharmaceutical compounds or compositions of the invention locally to thearea in need of treatment; this may be achieved by, for example, and notby way of limitation, local infusion during surgery, topicalapplication, e.g., in conjunction with a wound dressing after surgery,by injection, by means of a catheter, by means of a suppository, or bymeans of an implant, said implant being of a porous, non-porous, orgelatinous material, including membranes, such as sialastic membranes,or fibers. Preferably, when administering a protein, including anantibody, of the invention, care must be taken to use materials to whichthe protein does not absorb.

In another embodiment, the compound or composition can be delivered in avesicle, in particular a liposome (see Langer, Science 249:1527-1533(1990); Treat et al., in Liposomes in the Therapy of Infectious Diseaseand Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp.353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generallyibid.)

In yet another embodiment, the compound or composition can be deliveredin a controlled release system. In one embodiment, a pump may be used(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201 (1987);Buchwald et al., Surgery 88:507 (1980); Saudek et al., N. Engl. J. Med.321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer and Wise (eds.),CRC Press, Boca Raton, Fla. (1974); Controlled Drug Bioavailability,Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, NewYork (1984); Ranger and Peppas, J., Macromol. Sci. Rev. Macromol. Chem.23:61 (1983); see also Levy et al., Science 228:190 (1985); During etal., Ann. Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105(1989)). In yet another embodiment, a controlled release system can beplaced in proximity of the therapeutic target, i.e., the brain, thusrequiring only a fraction of the systemic dose (see, e.g., Goodson, inMedical Applications of Controlled Release, supra, vol. 2, pp. 115-138(1984)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

In a specific embodiment where the compound of the invention is anucleic acid encoding a protein, the nucleic acid can be administered invivo to promote expression of its encoded protein, by constructing it aspart of an appropriate nucleic acid expression vector and administeringit so that it becomes intracellular, e.g., by use of a retroviral vector(see U.S. Pat. No. 4,980,286), or by direct injection, or by use ofmicroparticle bombardment (e.g., a gene gun; Biolistic, Dupont), orcoating with lipids or cell-surface receptors or transfecting agents, orby administering it in linkage to a homeobox-like peptide which is knownto enter the nucleus (see e.g., Joliot et al., Proc. Natl. Acad. Sci.USA 88:1864-1868 (1991)), etc. Alternatively, a nucleic acid can beintroduced intracellularly and incorporated within host cell DNA forexpression, by homologous recombination.

The present invention also provides pharmaceutical compositions. Suchcompositions comprise a therapeutically effective amount of a compound,and a pharmaceutically acceptable carrier. In a specific embodiment, theterm “pharmaceutically acceptable” means approved by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans. The term “carrier” refers to a diluent,adjuvant, excipient, or vehicle with which the therapeutic isadministered. Such pharmaceutical carriers can be sterile liquids, suchas water and oils, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, sesameoil and the like. Water is a preferred carrier when the pharmaceuticalcomposition is administered intravenously. Saline solutions and aqueousdextrose and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. Suitable pharmaceuticalexcipients include starch, glucose, lactose, sucrose, gelatin, malt,rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate,talc, sodium chloride, dried skim milk, glycerol, propylene, glycol,water, ethanol and the like. The composition, if desired, can alsocontain minor amounts of wetting or emulsifying agents, or pH bufferingagents. These compositions can take the form of solutions, suspensions,emulsion, tablets, pills, capsules, powders, sustained-releaseformulations and the like. The composition can be formulated as asuppository, with traditional binders and carriers such astriglycerides. Oral formulation can include standard carriers such aspharmaceutical grades of mannitol, lactose, starch, magnesium stearate,sodium saccharine, cellulose, magnesium carbonate, etc. Examples ofsuitable pharmaceutical carriers are described in “Remington'sPharmaceutical Sciences” by E. W. Martin. Such compositions will containa therapeutically effective amount of the compound, preferably inpurified form, together with a suitable amount of carrier so as toprovide the form for proper administration to the patient. Theformulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated in accordancewith routine procedures as a pharmaceutical composition adapted forintravenous administration to human beings. Typically, compositions forintravenous administration are solutions in sterile isotonic aqueousbuffer. Where necessary, the composition may also include a solubilizingagent and a local anesthetic such as lignocaine to ease pain at the siteof the injection. Generally, the ingredients are supplied eitherseparately or mixed together in unit dosage form, for example, as a drylyophilized powder or water free concentrate in a hermetically sealedcontainer such as an ampoule or sachette indicating the quantity ofactive agent. Where the composition is to be administered by infusion,it can be dispensed with an infusion bottle containing sterilepharmaceutical grade water or saline. Where the composition isadministered by injection, an ampoule of sterile water for injection orsaline can be provided so that the ingredients may be mixed prior toadministration.

The compounds of the invention can be formulated as neutral or saltforms. Pharmaceutically acceptable salts include those formed withanions such as those derived from hydrochloric, phosphoric, acetic,oxalic, tartaric acids, etc., and those formed with cations such asthose derived from sodium, potassium, ammonium, calcium, ferrichydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol,histidine, procaine, etc.

The amount of the compound of the invention which will be effective inthe treatment, inhibition and prevention of a disease or disorderassociated with aberrant expression and/or activity of a polypeptide ofthe invention can be determined by standard clinical techniques. Inaddition, in vitro assays may optionally be employed to help identifyoptimal dosage ranges. The precise dose to be employed in theformulation will also depend on the route of administration, and theseriousness of the disease or disorder, and should be decided accordingto the judgment of the practitioner and each patient's circumstances.Effective doses may be extrapolated from dose-response curves derivedfrom in vitro or animal model test systems.

For antibodies, the dosage administered to a patient is typically 0.1mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosageadministered to a patient is between 0.1 mg/kg and 20 mg/kg of thepatient's body weight, more preferably 1 mg/kg to 10 mg/kg of thepatient's body weight. Generally, human antibodies have a longerhalf-life within the human body than antibodies from other species dueto the immune response to the foreign polypeptides. Thus, lower dosagesof human antibodies and less frequent administration is often possible.Further, the dosage and frequency of administration of antibodies of theinvention may be reduced by enhancing uptake and tissue penetration(e.g., into the brain) of the antibodies by modifications such as, forexample, lipidation.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Optionally associated withsuch container(s) can be a notice in the form prescribed by agovernmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, which notice reflects approvalby the agency of manufacture, use or sale for human administration.

Diagnosis and Imaging

Labeled antibodies, and derivatives and analogs thereof, whichspecifically bind to a polypeptide of interest can be used fordiagnostic purposes to detect, diagnose, or monitor diseases and/ordisorders associated with the aberrant expression and/or activity of apolypeptide of the invention. The invention provides for the detectionof aberrant expression of a polypeptide of interest, comprising (a)assaying the expression of the polypeptide of interest in cells or bodyfluid of an individual using one or more antibodies specific to thepolypeptide interest and (b) comparing the level of gene expression witha standard gene expression level, whereby an increase or decrease in theassayed polypeptide gene expression level compared to the standardexpression level is indicative of aberrant expression.

The invention provides a diagnostic assay for diagnosing a disorder,comprising (a) assaying the expression of the polypeptide of interest incells or body fluid of an individual using one or more antibodiesspecific to the polypeptide interest and (b) comparing the level of geneexpression with a standard gene expression level, whereby an increase ordecrease in the assayed polypeptide gene expression level compared tothe standard expression level is indicative of a particular disorder.With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Antibodies of the invention can be used to assay protein levels in abiological sample using classical immunohistological methods known tothose of skill in the art (e.g., see Jalkanen, et al., J. Cell. Biol.101:976-985 (1985); Jalkanen, et al., J. Cell. Biol. 105:3087-3096(1987)). Other antibody-based methods useful for detecting protein geneexpression include immunoassays, such as the enzyme linked immunosorbentassay (ELISA) and the radioimmunoassay (RIA). Suitable antibody assaylabels are known in the art and include enzyme labels, such as, glucoseoxidase; radioisotopes, such as iodine (¹³¹I, ¹²⁵I, ¹²³I, ¹²¹I), carbon(¹⁴C), sulfur (³⁵S), tritium (³H), indium (^(115m)In, ^(113m)In, ¹¹²In,¹¹¹In), and technetium (⁹⁹Tc, ^(99m)Tc), thallium (²⁰¹Ti), gallium(⁶⁸Ga, ⁶⁷Ga), palladium (¹⁰³Pd), molybdenum (⁹⁹Mo), xenon (¹³³Xe),fluorine (¹⁸F), ¹⁵³Sm, ¹⁷⁷Lu, ¹⁵⁹Gd, ¹⁴⁹Pm, ¹⁴⁰La, ¹⁷⁵Yb, ¹⁶⁶Ho, ⁹⁰Y,⁴⁷Sc, ¹⁸⁶Re, ¹⁸⁸Re, ¹⁴²Pr, ¹⁰⁵Rh, ⁹⁷Ru; luminescent labels, such asluminol; and fluorescent labels, such as fluorescein and rhodamine, andbiotin.

Techniques known in the art may be applied to label antibodies of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety).

One embodiment of the invention is the detection and diagnosis of adisease or disorder associated with aberrant expression of a polypeptideof interest in an animal, preferably a mammal and most preferably ahuman. In one embodiment, diagnosis comprises: (a) administering (forexample, parenterally, subcutaneously, or intraperitoneally) to asubject an effective amount of a labeled molecule which specificallybinds to the polypeptide of interest; (b) waiting for a time intervalfollowing the administering for permitting the labeled molecule topreferentially concentrate at sites in the subject where the polypeptideis expressed (and for unbound labeled molecule to be cleared tobackground level); (c) determining background level; and (d) detectingthe labeled molecule in the subject, such that detection of labeledmolecule above the background level indicates that the subject has aparticular disease or disorder associated with aberrant expression ofthe polypeptide of interest. Background level can be determined byvarious methods including, comparing the amount of labeled moleculedetected to a standard value previously determined for a particularsystem. As described herein, specific embodiments of the invention aredirected to the use of the antibodies of the invention to quantitate orqualitate concentrations of cells of B cell lineage or cells ofmonocytic lineage.

Also as described herein, antibodies of the invention may be used totreat, diagnose, or prognose an individual having an immunodeficiency.In a specific embodiment, antibodies of the invention are used to treat,diagnose, and/or prognose an individual having common variableimmunodeficiency disease (CVID) or a subset of this disease. In anotherembodiment, antibodies of the invention are used to diagnose, prognose,treat or prevent a disorder characterized by deficient serumimmunoglobulin production, recurrent infections, and/or immune systemdysfunction.

Also as described herein, antibodies of the invention may be used totreat, diagnose, or prognose an individual having an autoimmune diseaseor disorder. In a specific embodiment, antibodies of the invention areused to treat, diagnose, and/or prognose an individual having systemiclupus erythematosus, or a subset of the disease. In another specificembodiment, antibodies of the invention are used to treat, diagnoseand/or prognose an individual having rheumatoid arthritis, or a subsetof this disease.

It will be understood in the art that the size of the subject and theimaging system used will determine the quantity of imaging moiety neededto produce diagnostic images. In the case of a radioisotope moiety, fora human subject, the quantity of radioactivity injected will normallyrange from about 5 to 20 millicuries of ^(99m)Tc. The labeled antibodyor antibody fragment will then preferentially accumulate at the locationof cells which contain the specific protein. In vivo tumor imaging isdescribed in S. W. Burchiel et al., “Immunopharmacokinetics ofRadiolabeled Antibodies and Their Fragments.” (Chapter 13 in TumorImaging: The Radiochemical Detection of Cancer, S. W. Burchiel and B. A.Rhodes, eds., Masson Publishing Inc. (1982).

Depending on several variables, including the type of label used and themode of administration, the time interval following the administrationfor permitting the labeled molecule to preferentially concentrate atsites in the subject and for unbound labeled molecule to be cleared tobackground level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. Inanother embodiment the time interval following administration is 5 to 20days or 5 to 10 days.

In an embodiment, monitoring of the disease or disorder is carried outby repeating the method for diagnosing the disease or disease, forexample, one month after initial diagnosis, six months after initialdiagnosis, one year after initial diagnosis, etc.

Presence of the labeled molecule can be detected in the patient usingmethods known in the art for in vivo scanning. These methods depend uponthe type of label used. Skilled artisans will be able to determine theappropriate method for detecting a particular label. Methods and devicesthat may be used in the diagnostic methods of the invention include, butare not limited to, computed tomography (CT), whole body scan such asposition emission tomography (PET), magnetic resonance imaging (MRI),and sonography.

In a specific embodiment, the molecule is labeled with a radioisotopeand is detected in the patient using a radiation responsive surgicalinstrument (Thurston et al., U.S. Pat. No. 5,441,050). In anotherembodiment, the molecule is labeled with a fluorescent compound and isdetected in the patient using a fluorescence responsive scanninginstrument. In another embodiment, the molecule is labeled with apositron emitting metal and is detected in the patent using positronemission-tomography. In yet another embodiment, the molecule is labeledwith a paramagnetic label and is detected in a patient using magneticresonance imaging (MRI).

Kits

The present invention provides kits that can be used in the abovemethods. In one embodiment, a kit comprises an antibody of theinvention, preferably a purified antibody, in one or more containers. Ina specific embodiment, the kits of the present invention contain asubstantially isolated polypeptide comprising an epitope which isspecifically immunoreactive with an antibody included in the kit.Preferably, the kits of the present invention further comprise a controlantibody which does not react with the polypeptide of interest. Inanother specific embodiment, the kits of the present invention comprisetwo or more antibodies (monoclonal and/or polyclonal) that recognize thesame and/or different sequences or regions of the polypeptide of theinvention. In another specific embodiment, the kits of the presentinvention contain a means for detecting the binding of an antibody to apolypeptide of interest (e.g., the antibody may be conjugated to adetectable substrate such as a fluorescent compound, an enzymaticsubstrate, a radioactive compound or a luminescent compound, or a secondantibody which recognizes the first antibody may be conjugated to adetectable substrate).

In another specific embodiment of the present invention, the kit is adiagnostic kit for use in screening serum containing antibodies specificagainst proliferative and/or cancerous polynucleotides and polypeptides.Such a kit may include a control antibody that does not react with thepolypeptide of interest. Such a kit may include a substantially isolatedpolypeptide antigen comprising an epitope which is specificallyimmunoreactive with at least one anti-polypeptide antigen antibody.Further, such a kit includes means for detecting the binding of saidantibody to the antigen (e.g., the antibody may be conjugated to afluorescent compound such as fluorescein or rhodamine which can bedetected by flow cytometry). In specific embodiments, the kit mayinclude a recombinantly produced or chemically synthesized polypeptideantigen. The polypeptide antigen of the kit may also be attached to asolid support.

In a more specific embodiment the detecting means of the above-describedkit includes a solid support to which said polypeptide antigen isattached. Such a kit may also include a non-attached reporter-labeledanti-human antibody. In this embodiment, binding of the antibody to thepolypeptide antigen can be detected by binding of the saidreporter-labeled antibody.

In an additional embodiment, the invention includes a diagnostic kit foruse in screening serum containing antigens of the polypeptide of theinvention. The diagnostic kit includes a substantially isolated antibodyspecifically immunoreactive with polypeptide or polynucleotide antigens,and means for detecting the binding of the polynucleotide or polypeptideantigen to the antibody. In one embodiment, the antibody is attached toa solid support. In a specific embodiment, the antibody may be amonoclonal antibody. The detecting means of the kit may include asecond, labeled monoclonal antibody. Alternatively, or in addition, thedetecting means may include a labeled, competing antigen.

In one diagnostic configuration, test serum is reacted with a solidphase reagent having a surface-bound antigen obtained by the methods ofthe present invention. After binding with specific antigen antibody tothe reagent and removing unbound serum components by washing, thereagent is reacted with reporter-labeled anti-human antibody to bindreporter to the reagent in proportion to the amount of boundanti-antigen antibody on the solid support. The reagent is again washedto remove unbound labeled antibody, and the amount of reporterassociated with the reagent is determined. Typically, the reporter is anenzyme which is detected by incubating the solid phase in the presenceof a suitable fluorometric, luminescent or colorimetric substrate(Sigma, St. Louis, Mo.).

The solid surface reagent in the above assay is prepared by knowntechniques for attaching protein material to solid support material,such as polymeric beads, dip sticks, 96-well plate or filter material.These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment of the protein,typically through a free amine group, to a chemically reactive group onthe solid support, such as an activated carboxyl, hydroxyl, or aldehydegroup. Alternatively, streptavidin coated plates can be used inconjunction with biotinylated antigen(s).

Thus, the invention provides an assay system or kit for carrying outthis diagnostic method. The kit generally includes a support withsurface-bound recombinant antigens, and a reporter-labeled anti-humanantibody for detecting surface-bound anti-antigen antibody.

The invention further relates to antibodies which act as agonists orantagonists of the polypeptides of the present invention. For example,the present invention includes antibodies which disrupt thereceptor/ligand interactions with the polypeptides of the inventioneither partially or fully. Included are both receptor-specificantibodies and ligand-specific antibodies. Included arereceptor-specific antibodies which do not prevent ligand binding butprevent receptor activation. Receptor activation (i.e., signaling) maybe determined by techniques described herein or otherwise known in theart. Also included are receptor-specific antibodies which both preventligand binding and receptor activation. Likewise, included areneutralizing antibodies which bind the ligand and prevent binding of theligand to the receptor, as well as antibodies which bind the ligand,thereby preventing receptor activation, but do not prevent the ligandfrom binding the receptor. Further included are antibodies whichactivate the receptor. These antibodies may act as agonists for eitherall or less than all of the biological activities affected byligand-mediated receptor activation. The antibodies may be specified asagonists or antagonists for biological activities comprising specificactivities disclosed herein. Further included are antibodies that bindto D-SLAM irrespective of whether D-SLAM is bound to a D-SLAM Receptor.These antibodies act as D-SLAM agonists as reflected in an decrease incellular proliferation in response to binding of D-SLAM to a D-SLAMreceptor in the presence of these antibodies. The above antibodyagonists can be made using methods known in the art. See e.g., WO96/40281; U.S. Pat. No. 5,811,097; Deng, B. et al., Blood92(6):1981-1988 (1998); Chen, Z. et al., Cancer Res. 58(16):3668-3678(1998); Harrop, J. A. et al., J. Immunol. 161(4):1786-1794 (1998); Zhu,Z. et al., Cancer Res. 58(15):3209-3214 (1998); Yoon, D. Y. et al., J.Immunol. 160(7):3170-3179 (1998); Prat, M. et al., J. Cell. Sci.111(Pt2):237-247 (1998); Pitard, V. et al., J. Immunol. Methods205(2):177-190 (1997); Liautard, J. et al., Cytokinde 9(4):233-241(1997); Carlson, N. G. et al., J. Biol. Chem. 272(17):11295-11301(1997); Taryman, R. E. et al., Neuron 14(4):755-762 (1995); Muller, Y.A. et al., Structure 6(9):1153-1167 (1998); Bartunek, P. et al.,Cytokine 8(1):14-20 (1996) (said references incorporated by reference intheir entireties).

The invention encompasses antibodies that inhibit or reduce the abilityof D-SLAM to bind D-SLAM receptor in vitro and/or in vivo. In a specificembodiment, antibodies of the invention inhibit or reduce the ability ofD-SLAM to bind D-SLAM receptor in vitro. In another nonexclusivespecific embodiment, antibodies of the invention inhibit or reduce theability of D-SLAM to bind D-SLAM receptor in vivo. Such inhibition canbe assayed using techniques described herein or otherwise known in theart.

The invention also encompasses, antibodies that bind specifically toD-SLAM, but do not inhibit the ability of D-SLAM to bind D-SLAM receptorin vitro and/or in vivo. In a specific embodiment, antibodies of theinvention do not inhibit or reduce the ability of D-SLAM to bind D-SLAMreceptor in vitro. In another nonexclusive specific embodiment,antibodies of the invention do not inhibit or reduce the ability ofD-SLAM to bind D-SLAM receptor in vivo.

As described above, the invention encompasses antibodies that inhibit orreduce a D-SLAM-mediated biological activity in vitro and/or in vivo. Ina specific embodiment, antibodies of the invention inhibit or reduceD-SLAM-mediated inhibition of B cell proliferation in vitro. Suchinhibition can be assayed by routinely modifying B cell proliferationassays described herein or otherwise known in the art. In anothernonexclusive specific embodiment, antibodies of the invention inhibit orreduce D-SLAM-mediated inhibition of B cell proliferation in vivo.

Alternatively, the invention also encompasses, antibodies that bindspecifically to D-SLAM, but do not inhibit or reduce a D-SLAM-mediatedbiological activity in vitro and/or in vivo (e.g., inhibition of B cellproliferation). In a specific embodiment, antibodies of the invention donot inhibit or reduce a D-SLAM-mediated biological activity in vitro. Inanother non-exclusive embodiment, antibodies of the invention do notinhibit or reduce a D-SLAM-mediated biological activity in vivo.

As described above, the invention encompasses antibodies thatspecifically bind to the same epitope as at least one of the antibodiesspecifically referred to herein, in vitro and/or in vivo.

The antibodies of the invention also have uses as therapeutics and/orprophylactics which include, but are not limited to, in activatingmonocytes or blocking monocyte activation and/or killing specific celltypes that express the membrane bound form of D-SLAM on their cellsurfaces (e.g., to treat, prevent, and/or diagnose leukemias, lymphomas,rheumatoid arthritis, and other diseases or conditions associated withthese cell types). In a specific embodiment, the antibodies of theinvention fix complement. In other specific embodiments, as furtherdescribed herein, the antibodies of the invention (or fragments thereof)are associated with heterologous polypeptides or nucleic acids (e.g.toxins, such as, compounds that bind and activate endogenous cytotoxiceffecter systems, and radioisotopes; and cytotoxic prodrugs).

As discussed above, antibodies to the D-SLAM polypeptides of theinvention can, in turn, be utilized to generate anti-idiotype antibodiesthat “mimic” the D-SLAM, using techniques well known to those skilled inthe art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444 (1989), andNissinoff, J. Immunol. 147(8):2429-2438 (1991)). Such neutralizinganti-idiotypes or Fab fragments of such anti-idiotypes can be used intherapeutic regimens to neutralize D-SLAM ligand. For example, suchanti-idiotypic antibodies can be used to bind D-SLAM, or to bind D-SLAMreceptors on the surface of cells of B cell lineage, and thereby blockD-SLAM-mediated inhibition of B cell activation, proliferation, and/ordifferentiation.

Fusion Proteins

Any D-SLAM polypeptide can be used to generate fusion proteins. Forexample, the D-SLAM polypeptide, when fused to a second protein, can beused as an antigenic tag. Antibodies raised against the D-SLAMpolypeptide can be used to indirectly detect the second protein bybinding to the D-SLAM. Moreover, because secreted proteins targetcellular locations based on trafficking signals, the D-SLAM polypeptidescan be used as a targeting molecule once fused to other proteins.

Examples of domains that can be fused to D-SLAM polypeptides include notonly heterologous signal sequences, but also other heterologousfunctional regions. The fusion does not necessarily need to be direct,but may occur through linker sequences.

In certain preferred embodiments, D-SLAM proteins of the inventioncomprise, or alternatively consist of, fusion proteins wherein theD-SLAM polypeptides are those described above as m-n. In preferredembodiments, the application is directed to nucleic acid molecules atleast 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to thenucleic acid sequences encoding polypeptides having the amino acidsequence of the specific N- and C-terminal deletions recited herein.

Moreover, fusion proteins may also be engineered to improvecharacteristics of the D-SLAM polypeptide. For instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the N-terminus of the D-SLAM polypeptide to improve stability andpersistence during purification from the host cell or subsequenthandling and storage. Also, peptide moieties may be added to the D-SLAMpolypeptide to facilitate purification. Such regions may be removedprior to final preparation of the D-SLAM polypeptide. The addition ofpeptide moieties to facilitate handling of polypeptides are familiar androutine techniques in the art.

Moreover, D-SLAM polypeptides of the present invention, includingfragments thereof, and specifically epitope-bearing fragments may befused with heterologous polypeptide sequences. For example, thepolypeptides of the present invention may be fused with parts of theconstant domain of immunoglobulins (IgA, IgE, IgG, IgM) or portionsthereof (CH1, CH2, CH3, and any combination thereof, including bothentire domains and portions thereof), or albumin (including but notlimited to recombinant albumin), resulting in chimeric polypeptides.These fusion proteins facilitate purification and show an increasedhalf-life in vivo. One reported example describes chimeric proteinsconsisting of the first two domains of the human CD4-polypeptide andvarious domains of the constant regions of the heavy or light chains ofmammalian immunoglobulins. (EP A 394,827; Traunecker et al., Nature331:84-86 (1988).) Fusion proteins having disulfide-linked dimericstructures (due to the IgG) can also be more efficient in binding andneutralizing other molecules, than the monomeric secreted protein orprotein fragment alone. (Fountoulakis et al., J. Biochem. 270:3958-3964(1995).) Polynucleotides comprising or alternatively consisting ofnucleic acids which encode these fision proteins are also encompassed bythe invention.

Similarly, EP-A-O 464 533 (Canadian counterpart 2045869) disclosesfusion proteins comprising various portions of constant region ofimmunoglobulin molecules together with another human protein or partthereof. In many cases, the Fc part in a fusion protein is beneficial intherapy and diagnosis, and thus can result in, for example, improvedpharmacokinetic properties. (EP-A 0232 262.) Alternatively, deleting theFc part after the fusion protein has been expressed, detected, andpurified, would be desired. For example, the Fc portion may hindertherapy and diagnosis if the fusion protein is used as an antigen forimmunizations. In drug discovery, for example, human proteins, such ashIL-5, have been fused with Fc portions for the purpose ofhigh-throughput screening assays to identify antagonists of hIL-5. (See,D. Bennett et al., J. Molecular Recognition 8:52-58 (1995); K. Johansonet al., J. Biol. Chem. 270:9459-9471 (1995).)

Moreover, the D-SLAM polypeptides can be fused to marker sequences, suchas a peptide which facilitates purification of D-SLAM. In preferredembodiments, the marker amino acid sequence is a hexa-histidine peptide,such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 EtonAvenue, Chatsworth, Calif., 91311), among others, many of which arecommercially available. As described in Gentz et al., Proc. Natl. Acad.Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides forconvenient purification of the fusion protein. Another peptide taguseful for purification, the “HA” tag, corresponds to an epitope derivedfrom the influenza hemagglutinin protein. (Wilson et al., Cell 37:767(1984).)

Thus, any of these above fusions can be engineered using the D-SLAMpolynucleotides or the polypeptides.

Recombinant and Synthetic Production of D-SLAM

The present invention also relates to vectors containing the isolatedD-SLAM DNA molecules of the present invention, host cells which aregenetically engineered with the recombinant vectors, and the productionof polypeptides or fragments thereof by recombinant and synthetictechniques. The vector may be, for example, a phage, plasmid, viral, orretroviral vector. Retroviral vectors may be replication competent orreplication defective. In the latter case, viral propagation generallywill occur only in complementing host cells.

D-SLAM polynucleotides may be joined to a vector containing a selectablemarker for propagation in a host. Generally, a plasmid vector isintroduced in a precipitate, such as a calcium phosphate precipitate, orin a complex with a charged lipid. If the vector is a virus, it may bepackaged in vitro using an appropriate packaging cell line and thentransduced into host cells.

The D-SLAM polynucleotide insert should be operatively linked to anappropriate promoter, such as the phage lambda PL promoter, the E. colilac, trp, phoA and tac promoters, the SV40 early and late promoters andpromoters of retroviral LTRs, to name a few. Other suitable promoterswill be known to the skilled artisan. The expression constructs willfurther contain sites for transcription initiation, termination, and, inthe transcribed region, a ribosome binding site for translation. Thecoding portion of the transcripts expressed by the constructs willpreferably include a translation initiating codon at the beginning and atermination codon (UAA, UGA or UAG) appropriately positioned at the endof the polypeptide to be translated.

As indicated, the expression vectors will preferably include at leastone selectable marker. Such markers include dihydrofolate reductase,G418 or neomycin resistance for eukaryotic cell culture andtetracycline, kanamycin or ampicillin resistance genes for culturing inE. coli and other bacteria. Representative examples of appropriate hostsinclude, but are not limited to, bacterial cells, such as E. coli,Streptomyces and Salmonella typhimurium cells; fungal cells, such asyeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCCAccession No. 201178)); insect cells such as Drosophila S2 andSpodoptera Sf9 cells; animal cells such as CHO, COS, 293, and Bowesmelanoma cells; and plant cells. Appropriate culture mediums andconditions for the above-described host cells are known in the art.

Among vectors preferred for use in bacteria include pHE4-5 (ATCCAccession No. 209311; and variations thereof), pQE70, pQE60 and pQE-9,available from QIAGEN, Inc.; pBluescript vectors, Phagescript vectors,pNH8A, pNH16a, pNH18A, pNH46A, available from Stratagene CloningSystems, Inc.; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 availablefrom Pharmacia Biotech, Inc. Preferred expression vectors for use inyeast systems include, but are not limited to, pYES2, pYD1, pTEF1/Zeo,pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1,pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad,Calif.). Among preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44,pXT1 and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVLavailable from Pharmacia. Preferred expression vectors for use in yeastsystems include, but are not limited to pYES2, pYD1, pTEF1/Zeo,pYES2/GS, pPICZ, pGAPZ, pGAPZalph, pPIC9, pPIC3.5, pHIL-D2, pHIL-S1,pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad,Calif.). Other suitable vectors will be readily apparent to the skilledartisan. Other suitable vectors will be readily apparent to the skilledartisan.

In one embodiment, the yeast Pichia pastoris is used to express D-SLAMprotein in a eukaryotic system. Pichia pastoris is a methylotrophicyeast which can metabolize methanol as its sole carbon source. A mainstep in the methanol metabolization pathway is the oxidation of methanolto formaldehyde using O₂. This reaction is catalyzed by the enzymealcohol oxidase. In order to metabolize methanol as its sole carbonsource, Pichia pastoris must generate high levels of alcohol oxidasedue, in part, to the relatively low affinity of alcohol oxidase for O₂.Consequently, in a growth medium depending on methanol as a main carbonsource, the promoter region of one of the two alcohol oxidase genes(AOX1) is highly active. In the presence of methanol, alcohol oxidaseproduced from the AOX1 gene comprises up to approximately 30% of thetotal soluble protein in Pichia pastoris. See, Ellis, S. B., et al.,Mol. Cell. Biol. 5:1111-21 (1985); Koutz, P. J, et al., Yeast 5:167-77(1989); Tschopp, J. F., et al., Nucl. Acids Res. 15:3859-76 (1987).Thus, a heterologous coding sequence, such as, for example, a D-SLAMpolynucleotide of the present invention, under the transcriptionalregulation of all or part of the AOX1 regulatory sequence is expressedat exceptionally high levels in Pichia yeast grown in the presence ofmethanol.

In one example, the plasmid vector pPIC9K is used to express DNAencoding a D-SLAM polypeptide of the invention, as set forth herein, ina Pichea yeast system essentially as described in “Pichia Protocols:Methods in Molecular Biology,” D. R. Higgins and J. Cregg, eds. TheHumana Press, Totowa, N.J., 1998. This expression vector allowsexpression and secretion of a D-SLAM protein of the invention by virtueof the strong AOX1 promoter linked to the Pichia pastoris alkalinephosphatase (PHO) secretory signal peptide (i.e., leader) locatedupstream of a multiple cloning site.

Many other yeast vectors could be used in place of pPIC9K, such as,pYES2, pYD1, pTEF1/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9,pPIC3.5, pHIL-D2, pHIL-S1, pPIC3.5K, and PAO815, as one skilled in theart would readily appreciate, as long as the proposed expressionconstruct provides appropriately located signals for transcription,translation, secretion (if desired), and the like, including an in-frameAUG as required.

In one embodiment, high-level expression of a heterologous codingsequence, such as, for example, a D-SLAM polynucleotide of the presentinvention, may be achieved by cloning the heterologous polynucleotide ofthe invention into an expression vector such as, for example, pGAPZ orpGAPZalpha, and growing the yeast culture in the absence of methanol.

Introduction of the construct into the host cell can be effected bycalcium phosphate transfection, DEAE-dextran mediated transfection,cationic lipid-mediated transfection, electroporation, transduction,infection, or other methods. Such methods are described in many standardlaboratory manuals, such as Davis et al., Basic Methods In MolecularBiology (1986). It is specifically contemplated that D-SLAM polypeptidesmay in fact be expressed by a host cell lacking a recombinant vector.

D-SLAM polypeptides can be recovered and purified from recombinant cellcultures by well-known methods including ammonium sulfate or ethanolprecipitation, acid extraction, anion or cation exchange chromatography,phosphocellulose chromatography, hydrophobic interaction chromatography,affinity chromatography, hydroxylapatite chromatography and lectinchromatography. Most preferably, high performance liquid chromatography(“HPLC”) is employed for purification.

D-SLAM polypeptides, and preferably the secreted form, can also berecovered from: products purified from natural sources, including bodilyfluids, tissues and cells, whether directly isolated or cultured;products of chemical synthetic procedures; and products produced byrecombinant techniques from a prokaryotic or eukaryotic host, including,for example, bacterial, yeast, higher plant, insect, and mammaliancells. Depending upon the host employed in a recombinant productionprocedure, the D-SLAM polypeptides may be glycosylated or may benon-glycosylated. In addition, D-SLAM polypeptides may also include aninitial modified methionine residue, in some cases as a result ofhost-mediated processes. Thus, it is well known in the art that theN-terminal methionine encoded by the translation initiation codongenerally is removed with high efficiency from any protein aftertranslation in all eukaryotic cells. While the N-terminal methionine onmost proteins also is efficiently removed in most prokaryotes, for someproteins, this prokaryotic removal process is inefficient, depending onthe nature of the amino acid to which the N-terminal methionine iscovalently linked.

In addition to encompassing host cells containing the vector constructsdiscussed herein, the invention also encompasses primary, secondary, andimmortalized host cells of vertebrate origin, particularly mammalianorigin, that have been engineered to delete or replace endogenousgenetic material (e.g., D-SLAM coding sequence), and/or to includegenetic material (e.g., heterologous polynucleotide sequences) that isoperably associated with D-SLAM polynucleotides of the invention, andwhich activates, alters, and/or amplifies endogenous D-SLAMpolynucleotides. For example, techniques known in the art may be used tooperably associate heterologous control regions (e.g., promoter and/orenhancer) and endogenous D-SLAM polynucleotide sequences via homologousrecombination (see, e.g., U.S. Pat. No. 5,641,670, issued Jun. 24, 1997;International Publication No. WO 96/29411, published Sep. 26, 1996;International Publication No. WO 94/12650, published Aug. 4, 1994;Koller et al., Proc. Natl. Acad. Sci. USA 86:8932-8935 (1989); andZijlstra et al., Nature 342:435-438 (1989), the disclosures of each ofwhich are incorporated by reference in their entireties).

In addition, polypeptides of the invention can be chemically synthesizedusing techniques known in the art (e.g., see Creighton, 1983, Proteins:Structures and Molecular Principles, W. H. Freeman & Co., N.Y., andHunkapiller, M., et al., 1984, Nature 310:105-111). For example, apeptide corresponding to a fragment of the D-SLAM polypeptides of theinvention can be synthesized by use of a peptide synthesizer.Furthermore, if desired, nonclassical amino acids or chemical amino acidanalogs can be introduced as a substitution or addition into the D-SLAMpolynucleotide sequence. Non-classical amino acids include, but are notlimited to, to the D-isomers of the common amino acids,2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid,Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib,2-amino isobutyric acid, 3-amino propionic acid, omithine, norleucine,norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline,cysteic acid, t-butylglycine, t-butylalanine, phenylglycine,cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acidssuch as b-methyl amino acids, Ca-methyl amino acids, Na-methyl aminoacids, and amino acid analogs in general. Furthermore, the amino acidcan be D (dextrorotary) or L (levorotary).

The invention encompasses D-SLAM polypeptides which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule or other cellular ligand, etc. Any of numerous chemicalmodifications may be carried out by known techniques, including but notlimited, to specific chemical cleavage by cyanogen bromide, trypsin,chymotrypsin, papain, V8 protease, NaBH₄; acetylation, formylation,oxidation, reduction; metabolic synthesis in the presence oftunicamycin; etc.

Additional post-translational modifications encompassed by the inventioninclude, for example, e.g., N-linked or O-linked carbohydrate chains,processing of N-terminal or C-terminal ends), attachment of chemicalmoieties to the amino acid backbone, chemical modifications of N-linkedor O-linked carbohydrate chains, and addition or deletion of anN-terminal methionine residue as a result of procaryotic host cellexpression. The polypeptides may also be modified with a detectablelabel, such as an enzymatic, fluorescent, isotopic or affinity label toallow for detection and isolation of the protein.

Also provided by the invention are chemically modified derivatives ofD-SLAM which may provide additional advantages such as increasedsolubility, stability and circulating time of the polypeptide, ordecreased immunogenicity (see U.S. Pat. No. 4,179,337). The chemicalmoieties for derivitization may be selected from water soluble polymerssuch as polyethylene glycol, ethylene glycol/propylene glycolcopolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and thelike. The polypeptides may be modified at random positions within themolecule, or at predetermined positions within the molecule and mayinclude one, two, three or more attached chemical moieties.

The polymer may be of any molecular weight, and may be branched orunbranched. For polyethylene glycol, the preferred molecular weight isbetween about 1 kDa and about 100 kDa (the term “about” indicating thatin preparations of polyethylene glycol, some molecules will weigh more,some less, than the stated molecular weight) for ease in handling andmanufacturing. Other sizes may be used, depending on the desiredtherapeutic profile (e.g., the duration of sustained release desired,the effects, if any on biological activity, the ease in handling, or thelack of antigenicity and other known effects of the polyethylene glycolto a therapeutic protein or analog).

The polyethylene glycol molecules (or other chemical moieties) should beattached to the protein with consideration of effects on functional orantigenic domains of the protein. There are a number of attachmentmethods available to those skilled in the art, e.g., EP 0 401 384,herein incorporated by reference (coupling PEG to G-CSF), see also Maliket al., Exp. Hematol. 20:1028-1035 (1992) (reporting pegylation ofGM-CSF using tresyl chloride). For example, polyethylene glycol may becovalently bound through amino acid residues via a reactive group, suchas, a free amino or carboxyl group. Reactive groups are those to whichan activated polyethylene glycol molecule may be bound. The amino acidresidues having a free amino group may include lysine residues and theN-terminal amino acid residues; those having a free carboxyl group mayinclude aspartic acid residues glutamic acid residues and the C-terminalamino acid residue. Sulfhydryl groups may also be used as a reactivegroup for attaching the polyethylene glycol molecules. Preferred fortherapeutic purposes is attachment at an amino group, such as attachmentat the N-terminus or lysine group.

One may specifically desire proteins chemically modified at theN-terminus. Using polyethylene glycol as an illustration of the presentcomposition, one may select from a variety of polyethylene glycolmolecules (by molecular weight, branching, etc.), the proportion ofpolyethylene glycol molecules to protein (or peptide) molecules in thereaction mix, the type of pegylation reaction to be performed, and themethod of obtaining the selected N-terminally pegylated protein. Themethod of obtaining the N-terminally pegylated preparation (i.e.,separating this moiety from other monopegylated moieties if necessary)may be by purification of the N-terminally pegylated material from apopulation of pegylated protein molecules. Selective proteins chemicallymodified at the N-terminus modification may be accomplished by reductivealkylation which exploits differential reactivity of different types ofprimary amino groups (lysine versus the N-terminal) available forderivatization in a particular protein. Under the appropriate reactionconditions, substantially selective derivatization of the protein at theN-terminus with a carbonyl group containing polymer is achieved.

The D-SLAM polypeptides of the invention may be in monomers or multimers(i.e., dimers, trimers, tetramers and higher multimers). Accordingly,the present invention relates to monomers and multimers of the D-SLAMpolypeptides of the invention, their preparation, and compositions(preferably, pharmaceutical compositions) containing them. In specificembodiments, the polypeptides of the invention are monomers, dimers,trimers or tetramers. In additional embodiments, the multimers of theinvention are at least dimers, at least trimers, or at least tetramers.

Multimers encompassed by the invention may be homomers or heteromers. Asused herein, the term homomer, refers to a multimer containing onlyD-SLAM polypeptides of the invention (including D-SLAM fragments,variants, splice variants, and fusion proteins, as described herein).These homomers may contain D-SLAM polypeptides having identical ordifferent amino acid sequences. In a specific embodiment, a homomer ofthe invention is a multimer containing only D-SLAM polypeptides havingan identical amino acid sequence. In another specific embodiment, ahomomer of the invention is a multimer containing D-SLAM polypeptideshaving different amino acid sequences. In specific embodiments, themultimer of the invention is a homodimer (e.g., containing D-SLAMpolypeptides having identical or different amino acid sequences) or ahomotrimer (e.g., containing D-SLAM polypeptides having identical and/ordifferent amino acid sequences). In additional embodiments, thehomomeric multimer of the invention is at least a homodimer, at least ahomotrimer, or at least a homotetramer.

As used herein, the term heteromer refers to a multimer containing oneor more heterologous polypeptides (i.e., polypeptides of differentproteins) in addition to the D-SLAM polypeptides of the invention. In aspecific embodiment, the multimer of the invention is a heterodimer, aheterotrimer, or a heterotetramer. In additional embodiments, thehomomeric multimer of the invention is at least a homodimer, at least ahomotrimer, or at least a homotetramer.

Multimers of the invention may be the result of hydrophobic,hydrophilic, ionic and/or covalent associations and/or may be indirectlylinked, by for example, liposome formation. Thus, in one embodiment,multimers of the invention, such as, for example, homodimers orhomotrimers, are formed when polypeptides of the invention contact oneanother in solution. In another embodiment, heteromultimers of theinvention, such as, for example, heterotrimers or heterotetramers, areformed when polypeptides of the invention contact antibodies to thepolypeptides of the invention (including antibodies to the heterologouspolypeptide sequence in a fusion protein of the invention) in solution.In other embodiments, multimers of the invention are formed by covalentassociations with and/or between the D-SLAM polypeptides of theinvention. Such covalent associations may involve one or more amino acidresidues contained in the polypeptide sequence (e.g., that recited inSEQ ID NO:2, or contained in the polypeptide encoded by the cloneHDPJO39). In one instance, the covalent associations are cross-linkingbetween cysteine residues located within the polypeptide sequences whichinteract in the native (i.e., naturally occurring) polypeptide. Inanother instance, the covalent associations are the consequence ofchemical or recombinant manipulation. Alternatively, such covalentassociations may involve one or more amino acid residues contained inthe heterologous polypeptide sequence in a D-SLAM fusion protein. In oneexample, covalent associations are between the heterologous sequencecontained in a fusion protein of the invention (see, e.g., U.S. Pat. No.5,478,925). In a specific example, the covalent associations are betweenthe heterologous sequence contained in a D-SLAM-Fc fusion protein of theinvention (as described herein). In another specific example, covalentassociations of fusion proteins of the invention are betweenheterologous polypeptide sequence from another Secreted LymphocyteActivation Molecule (SLAM) family member that is capable of formingcovalently associated multimers, such as for example, oseteoprotegerin(see, e.g., International Publication No. WO 98/49305, the contents ofwhich are herein incorporated by reference in its entirety). In anotherembodiment, two or more polypeptides of the invention are joined throughpeptide linkers. Examples include those peptide linkers described inU.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteinscomprising multiple polypeptides of the invention separated by peptidelinkers may be produced using conventional recombinant DNA technology.

Another method for preparing multimer polypeptides of the inventioninvolves use of polypeptides of the invention fused to a leucine zipperor isoleucine zipper polypeptide sequence. Leucine zipper and isoleucinezipper domains are polypeptides that promote multimerization of theproteins in which they are found. Leucine zippers were originallyidentified in several DNA-binding proteins (Landschulz et al., Science240:1759, (1988)), and have since been found in a variety of differentproteins. Among the known leucine zippers are naturally occurringpeptides and derivatives thereof that dimerize or trimerize. Examples ofleucine zipper domains suitable for producing soluble multimericproteins of the invention are those described in PCT application WO94/10308, hereby incorporated by reference. Recombinant fusion proteinscomprising a polypeptide of the invention fused to a polypeptidesequence that dimerizes or trimerizes in solution are expressed insuitable host cells, and the resulting soluble multimeric fusion proteinis recovered from the culture supernatant using techniques known in theart.

Trimeric polypeptides of the invention may offer the advantage ofenhanced biological activity. Preferred leucine zipper moieties andisoleucine moieties are those that preferentially form trimers. Oneexample is a leucine zipper derived from lung surfactant protein D(SPD), as described in Hoppe et al. (FEBS Letters 344:191, (1994)) andin U.S. patent application Ser. No. 08/446,922, hereby incorporated byreference. Other peptides derived from naturally occurring trimericproteins may be employed in preparing trimeric polypeptides of theinvention.

In another example, proteins of the invention are associated byinteractions between Flag® polypeptide sequence contained in fusionproteins of the invention containing Flag® polypeptide sequence. In afurther embodiment, associations proteins of the invention areassociated by interactions between heterologous polypeptide sequencecontained in Flag® fusion proteins of the invention and anti-Flag®antibody.

The multimers of the invention may be generated using chemicaltechniques known in the art. For example, polypeptides desired to becontained in the multimers of the invention may be chemicallycross-linked using linker molecules and linker molecule lengthoptimization techniques known in the art (see, e.g., U.S. Pat. No.5,478,925, which is herein incorporated by reference in its entirety).Additionally, multimers of the invention may be generated usingtechniques known in the art to form one or more inter-moleculecross-links between the cysteine residues located within the sequence ofthe polypeptides desired to be contained in the multimer (see, e.g.,U.S. Pat. No. 5,478,925, which is herein incorporated by reference inits entirety). Further, polypeptides of the invention may be routinelymodified by the addition of cysteine or biotin to the C terminus orN-terminus of the polypeptide and techniques known in the art may beapplied to generate multimers containing one or more of these modifiedpolypeptides (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety). Additionally, techniquesknown in the art may be applied to generate liposomes containing thepolypeptide components desired to be contained in the multimer of theinvention (see, e.g., U.S. Pat. No. 5,478,925, which is hereinincorporated by reference in its entirety).

Alternatively, multimers of the invention may be generated using geneticengineering techniques known in the art. In one embodiment, polypeptidescontained in multimers of the invention are produced recombinantly usingfusion protein technology described herein or otherwise known in the art(see, e.g., U.S. Pat. No. 5,478,925, which is herein incorporated byreference in its entirety). In a specific embodiment, polynucleotidescoding for a homodimer of the invention are generated by ligating apolynucleotide sequence encoding a polypeptide of the invention to asequence encoding a linker polypeptide and then further to a syntheticpolynucleotide encoding the translated product of the polypeptide in thereverse orientation from the original C-terminus to the N-terminus(lacking the leader sequence) (see, e.g., U.S. Pat. No. 5,478,925, whichis herein incorporated by reference in its entirety). In anotherembodiment, recombinant techniques described herein or otherwise knownin the art are applied to generate recombinant polypeptides of theinvention which contain a transmembrane domain (or hydrophobic or signalpeptide) and which can be incorporated by membrane reconstitutiontechniques into liposomes (see, e.g., U.S. Pat. No. 5,478,925, which isherein incorporated by reference in its entirety).

Uses of the D-SLAM Polynucleotides

The D-SLAM polynucleotides identified herein can be used in numerousways as reagents. The following description should be consideredexemplary and utilizes known techniques.

There exists an ongoing need to identify new chromosome markers, sincefew chromosome marking reagents, based on actual sequence data (repeatpolymorphisms), are presently available.

Briefly, sequences can be mapped to chromosomes by preparing PCR primers(preferably 15-25 bp) from the sequences shown in SEQ ID NO:1. Primerscan be selected using computer analysis so that primers do not span morethan one predicted exon in the genomic DNA. These primers are then usedfor PCR screening of somatic cell hybrids containing individual humanchromosomes. Only those hybrids containing the human D-SLAM genecorresponding to the SEQ ID NO:1 will yield an amplified fragment.

Similarly, somatic hybrids provide a rapid method of PCR mapping thepolynucleotides to particular chromosomes. Three or more clones can beassigned per day using a single thermal cycler. Moreover,sublocalization of the D-SLAM polynucleotides can be achieved withpanels of specific chromosome fragments. Other gene mapping strategiesthat can be used include in situ hybridization, prescreening withlabeled flow-sorted chromosomes, and preselection by hybridization toconstruct chromosome specific-cDNA libraries.

Precise chromosomal location of the D-SLAM polynucleotides can also beachieved using fluorescence in situ hybridization (FISH) of a metaphasechromosomal spread. This technique uses polynucleotides as short as 500or 600 bases; however, polynucleotides 2,000-4,000 bp are preferred. Fora review of this technique, see Verma et al., “Human Chromosomes: aManual of Basic Techniques,” Pergamon Press, New York (1988).

For chromosome mapping, the D-SLAM polynucleotides can be usedindividually (to mark a single chromosome or a single site on thatchromosome) or in panels (for marking multiple sites and/or multiplechromosomes). Preferred polynucleotides correspond to the noncodingregions of the cDNAs because the coding sequences are more likelyconserved within gene families, thus increasing the chance of crosshybridization during chromosomal mapping.

Once a polynucleotide has been mapped to a precise chromosomal location,the physical position of the polynucleotide can be used in linkageanalysis. Linkage analysis establishes coinheritance between achromosomal location and presentation of a particular disease. (Diseasemapping data are found, for example, in V. McKusick, MendelianInheritance in Man (available on line through Johns Hopkins UniversityWelch Medical Library).) Assuming 1 megabase mapping resolution and onegene per 20 kb, a cDNA precisely localized to a chromosomal regionassociated with the disease could be one of 50-500 potential causativegenes.

Thus, once coinheritance is established, differences in the D-SLAMpolynucleotide and the corresponding gene between affected andunaffected individuals can be examined. First, visible structuralalterations in the chromosomes, such as deletions or translocations, areexamined in chromosome spreads or by PCR. If no structural alterationsexist, the presence of point mutations are ascertained. Mutationsobserved in some or all affected individuals, but not in normalindividuals, indicates that the mutation may cause the disease. However,complete sequencing of the D-SLAM polypeptide and the corresponding genefrom several normal individuals is required to distinguish the mutationfrom a polymorphism. If a new polymorphism is identified, thispolymorphic polypeptide can be used for further linkage analysis.

Furthermore, increased or decreased expression of the gene in affectedindividuals as compared to unaffected individuals can be assessed usingD-SLAM polynucleotides. Any of these alterations (altered expression,chromosomal rearrangement, or mutation) can be used as a diagnostic orprognostic marker.

In addition to the foregoing, a D-SLAM polynucleotide can be used tocontrol gene expression through triple helix formation or antisense DNAor RNA. Both methods rely on binding of the polynucleotide to DNA orRNA. For these techniques, preferred polynucleotides are usually 20 to40 bases in length and complementary to either the region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. Acids Res.6:3073 (1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1360 (1991)) or to the mRNA itself (antisense—Okano, J.Neurochem. 56:560 (1991); Oligodeoxy-nucleotides as Antisense Inhibitorsof Gene Expression, CRC Press, Boca Raton, Fla. (1988).) Triple helixformation optimally results in a shut-off of RNA transcription from DNA,while antisense RNA hybridization blocks translation of an mRNA moleculeinto polypeptide. Both techniques are effective in model systems, andthe information disclosed herein can be used to design antisense ortriple helix polynucleotides in an effort to treat, diagnose, detect,and/or prevent disease.

D-SLAM polynucleotides are also useful in gene therapy. One goal of genetherapy is to insert a normal gene into an organism having a defectivegene, in an effort to correct the genetic defect. D-SLAM offers a meansof targeting such genetic defects in a highly accurate manner. Anothergoal is to insert a new gene that was not present in the host genome,thereby producing a new trait in the host cell.

The D-SLAM polynucleotides are also useful for identifying individualsfrom minute biological samples. The United States military, for example,is considering the use of restriction fragment length polymorphism(RFLP) for identification of its personnel. In this technique, anindividual's genomic DNA is digested with one or more restrictionenzymes, and probed on a Southern blot to yield unique bands foridentifying personnel. This method does not suffer from the currentlimitations of “Dog Tags” which can be lost, switched, or stolen, makingpositive identification difficult. The D-SLAM polynucleotides can beused as additional DNA markers for RFLP.

The D-SLAM polynucleotides can also be used as an alternative to RFLP,by determining the actual base-by-base DNA sequence of selected portionsof an individual's genome. These sequences can be used to prepare PCRprimers for amplifying and isolating such selected DNA, which can thenbe sequenced. Using this technique, individuals can be identifiedbecause each individual will have a unique set of DNA sequences. Once anunique ID database is established for an individual, positiveidentification of that individual, living or dead, can be made fromextremely small tissue samples.

Forensic biology also benefits from using DNA-based identificationtechniques as disclosed herein. DNA sequences taken from very smallbiological samples such as tissues, e.g., hair or skin, or body fluids,e.g., blood, saliva, semen, etc., can be amplified using PCR. In oneprior art technique, gene sequences amplified from polymorphic loci,such as DQa class II HLA gene, are used in forensic biology to identifyindividuals. (Erlich, H., PCR Technology, Freeman and Co. (1992).) Oncethese specific polymorphic loci are amplified, they are digested withone or more restriction enzymes, yielding an identifying set of bands ona Southern blot probed with DNA corresponding to the DQa class II HLAgene. Similarly, D-SLAM polynucleotides can be used as polymorphicmarkers for forensic purposes.

There is also a need for reagents capable of identifying the source of aparticular tissue. Such need arises, for example, in forensics whenpresented with tissue of unknown origin. Appropriate reagents cancomprise, or alternatively consist of, for example, DNA probes orprimers specific to particular tissue prepared from D-SLAM sequences.Panels of such reagents can identify tissue by species and/or by organtype. In a similar fashion, these reagents can be used to screen tissuecultures for contamination.

Because D-SLAM is found expressed in dendritic cells, T cell lymphoma,lymph node, spleen, thymus, small intestine, and uterus, D-SLAMpolynucleotides are useful as hybridization probes for differentialidentification of the tissue(s) or cell type(s) present in a biologicalsample. Similarly, polypeptides and antibodies directed to D-SLAMpolypeptides are useful to provide immunological probes for differentialidentification of the tissue(s) or cell type(s). In addition, for anumber of disorders of the above tissues or cells, particularly of theimmune system, significantly higher or lower levels of D-SLAM geneexpression may be detected in certain tissues (e.g., cancerous andwounded tissues) or bodily fluids (e.g., serum, plasma, urine, synovialfluid or spinal fluid) taken from an individual having such a disorder,relative to a “standard” D-SLAM gene expression level, i.e., the D-SLAMexpression level in healthy tissue from an individual not having theimmune system disorder.

Thus, the invention provides a diagnostic method of a disorder, whichinvolves: (a) assaying D-SLAM gene expression level in cells or bodyfluid of an individual; (b) comparing the D-SLAM gene expression levelwith a standard D-SLAM gene expression level, whereby an increase ordecrease in the assayed D-SLAM gene expression level compared to thestandard expression level is indicative of disorder in the immunesystem.

In the very least, the D-SLAM polynucleotides can be used as molecularweight markers on Southern gels, as diagnostic probes for the presenceof a specific mRNA in a particular cell type, as a probe to“subtract-out” known sequences in the process of discovering novelpolynucleotides, for selecting and making oligomers for attachment to a“gene chip” or other support, to raise anti-DNA antibodies using DNAimmunization techniques, and as an antigen to elicit an immune response.

Uses of D-SLAM Polypeptides

D-SLAM polypeptides can be used in numerous ways. The followingdescription should be considered exemplary and utilizes knowntechniques.

D-SLAM polypeptides can be used to assay protein levels in a biologicalsample using antibody-based techniques. For example, protein expressionin tissues can be studied with classical immunohistological methods.(Jalkanen, M., et al., J. Cell. Biol. 101:976-985 (1985); Jalkanen, M.,et al., J. Cell. Biol. 105:3087-3096 (1987).) Other antibody-basedmethods useful for detecting protein gene expression includeimmunoassays, such as the enzyme linked immunosorbent assay (ELISA) andthe radioimmunoassay (RIA). Suitable antibody assay labels are known inthe art and include enzyme labels, such as, glucose oxidase, andradioisotopes, such as iodine (¹²⁵I, ¹²¹I), carbon (¹⁴C), sulfur (³⁵S),tritium (³H), indium (¹¹²In), and technetium (^(99m)Tc), and fluorescentlabels, such as fluorescein and rhodamine, and biotin.

In addition to assaying secreted protein levels in a biological sample,proteins can also be detected in vivo by imaging. Antibody labels ormarkers for in vivo imaging of protein include those detectable byX-radiography, NMR or ESR. For X-radiography, suitable labels includeradioisotopes such as barium or cesium, which emit detectable radiationbut are not overtly harmful to the subject. Suitable markers for NMR andESR include those with a detectable characteristic spin, such asdeuterium, which may be incorporated into the antibody by labeling ofnutrients for the relevant hybridoma.

A protein-specific antibody or antibody fragment which has been labeledwith an appropriate detectable imaging moiety, such as a radioisotope(for example, 131I, 112In, 99mTc), a radio-opaque substance, or amaterial detectable by nuclear magnetic resonance, is introduced (forexample, parenterally, subcutaneously, or intraperitoneally) into themammal. It will be understood in the art that the size of the subjectand the imaging system used will determine the quantity of imagingmoiety needed to produce diagnostic images. In the case of aradioisotope moiety, for a human subject, the quantity of radioactivityinjected will normally range from about 5 to 20 millicuries of 99mTc.The labeled antibody or antibody fragment will then preferentiallyaccumulate at the location of cells which contain the specific protein.In vivo tumor imaging is described in S. W. Burchiel et al.,“Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments.”(Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982).)

Thus, the invention provides a diagnostic method of a disorder, whichinvolves (a) assaying the expression of D-SLAM polypeptide in cells orbody fluid of an individual; (b) comparing the level of gene expressionwith a standard gene expression level, whereby an increase or decreasein the assayed D-SLAM polypeptide gene expression level compared to thestandard expression level is indicative of a disorder.

Moreover, D-SLAM polypeptides can be used to treat, diagnose, detect,and/or prevent disease. For example, patients can be administered D-SLAMpolypeptides in an effort to replace absent or decreased levels of theD-SLAM polypeptide (e.g., insulin), to supplement absent or decreasedlevels of a different polypeptide (e.g., hemoglobin S for hemoglobin B),to inhibit the activity of a polypeptide (e.g., an oncogene), toactivate the activity of a polypeptide (e.g., by binding to a receptor),to reduce the activity of a membrane bound receptor by competing with itfor free ligand (e.g., soluble TNF receptors used in reducinginflammation), or to bring about a desired response (e.g., blood vesselgrowth).

Similarly, antibodies directed to D-SLAM polypeptides can also be usedto treat, diagnose, detect, and/or prevent disease. For example,administration of an antibody directed to a D-SLAM polypeptide can bindand reduce overproduction of the polypeptide. Similarly, administrationof an antibody can activate the polypeptide, such as by binding to apolypeptide bound to a membrane (receptor).

At the very least, the D-SLAM polypeptides can be used as molecularweight markers on SDS-PAGE gels or on molecular sieve gel filtrationcolumns using methods well known to those of skill in the art. D-SLAMpolypeptides can also be used to raise antibodies, which in turn areused to measure protein expression from a recombinant cell, as a way ofassessing transformation of the host cell. Moreover, D-SLAM polypeptidescan be used to test the following biological activities.

Gene Therapy Methods

Another aspect of the present invention is to gene therapy methods fortreating, diagnosing, detecting, and/or preventing disorders, diseasesand/or conditions. The gene therapy methods relate to the introductionof nucleic acid (DNA, RNA and antisense DNA or RNA) sequences into ananimal to achieve expression of the D-SLAM polypeptide of the presentinvention. This method requires a polynucleotide which codes for aD-SLAM polypeptide operatively linked to a promoter and any othergenetic elements necessary for the expression of the polypeptide by thetarget tissue. Such gene therapy and delivery techniques are known inthe art, see, for example, WO90/11092, which is herein incorporated byreference.

Thus, for example, cells from a patient may be engineered with apolynucleotide (DNA or RNA) comprising a promoter operably linked to aD-SLAM polynucleotide ex vivo, with the engineered cells then beingprovided to a patient to be treated (therapeutically and/orprophylactically) and/or diagnosed with the polypeptide. Such methodsare well-known in the art. For example, see Belldegrun, A., et al., J.Natl. Cancer Inst. 85: 207-216 (1993); Ferrantini, M. et al., CancerResearch 53: 1107-1112 (1993); Ferrantini, M. et al., J. Immunology 153:4604-4615 (1994); Kaido, T., et al., Int. J. Cancer 60: 221-229 (1995);Ogura, H., et al., Cancer Research 50: 5102-5106 (1990); Santodonato,L., et al., Human Gene Therapy 7:1-10 (1996); Santodonato, L., et al.,Gene Therapy 4:1246-1255 (1997); and Zhang, J.-F. et al., Cancer GeneTherapy 3: 31-38 (1996)), which are herein incorporated by reference. Inone embodiment, the cells which are engineered are arterial cells. Thearterial cells may be reintroduced into the patient through directinjection to the artery, the tissues surrounding the artery, or throughcatheter injection.

As discussed in more detail below, the D-SLAM polynucleotide constructscan be delivered by any method that delivers injectable materials to thecells of an animal, such as, injection into the interstitial space oftissues (heart, muscle, skin, lung, liver, and the like). The D-SLAMpolynucleotide constructs may be delivered in a pharmaceuticallyacceptable liquid or aqueous carrier.

In one embodiment, the D-SLAM polynucleotide is delivered as a nakedpolynucleotide. The term “naked” polynucleotide, DNA or RNA refers tosequences that are free from any delivery vehicle that acts to assist,promote or facilitate entry into the cell, including viral sequences,viral particles, liposome formulations, lipofectin or precipitatingagents and the like. However, the D-SLAM polynucleotides can also bedelivered in liposome formulations and lipofectin formulations and thelike can be prepared by methods well known to those skilled in the art.Such methods are described, for example, in U.S. Pat. Nos. 5,593,972,5,589,466, and 5,580,859, which are herein incorporated by reference.

The D-SLAM polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication.Appropriate vectors include pWLNEO, pSV2CAT, pOG44, pXT1 and pSGavailable from Stratagene; pSVK3, pBPV, pMSG and pSVL available fromPharmacia; and pEF11V5, pcDNA3.1, and pRc/CMV2 available fromInvitrogen. Other suitable vectors will be readily apparent to theskilled artisan.

Any strong promoter known to those skilled in the art can be used fordriving the expression of D-SLAM DNA. Suitable promoters includeadenoviral promoters, such as the adenoviral major late promoter; orheterologous promoters, such as the cytomegalovirus (CMV) promoter; therespiratory syncytial virus (RSV) promoter; inducible promoters, such asthe MMT promoter, the metallothionein promoter; heat shock promoters;the albumin promoter; the ApoAI promoter; human globin promoters; viralthymidine kinase promoters, such as the Herpes Simplex thymidine kinasepromoter; retroviral LTRs; the b-actin promoter; and human growthhormone promoters. The promoter also may be the native promoter forD-SLAM.

Unlike other gene therapy techniques, one major advantage of introducingnaked nucleic acid sequences into target cells is the transitory natureof the polynucleotide synthesis in the cells. Studies have shown thatnon-replicating DNA sequences can be introduced into cells to provideproduction of the desired polypeptide for periods of up to six months.

The D-SLAM polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellular,fluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked acid sequence injection, an effective dosage amount of DNAor RNA will be in the range of from about 0.05 mg/kg body weight toabout 50 mg/kg body weight. Preferably the dosage will be from about0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05 mg/kgto about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated (therapeutically and/orprophylactically) and the route of administration.

The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked D-SLAM DNAconstructs can be delivered to arteries during angioplasty by thecatheter used in the procedure.

The naked polynucleotides are delivered by any method known in the art,including, but not limited to, direct needle injection at the deliverysite, intravenous injection, topical administration, catheter infusion,and so-called “gene guns”. These delivery methods are known in the art.

As is evidenced in the Examples, naked D-SLAM nucleic acid sequences canbe administered in vivo results in the successful expression of D-SLAMpolypeptide in the femoral arteries of rabbits.

The constructs may also be delivered with delivery vehicles such asviral sequences, viral particles, liposome formulations, lipofectin,precipitating agents, etc. Such methods of delivery are known in theart.

In certain embodiments, the D-SLAM polynucleotide constructs arecomplexed in a liposome preparation. Liposomal preparations for use inthe instant invention include cationic (positively charged), anionic(negatively charged) and neutral preparations. However, cationicliposomes are particularly preferred because a tight charge complex canbe formed between the cationic liposome and the polyanionic nucleicacid. Cationic liposomes have been shown to mediate intracellulardelivery of plasmid DNA (Felgner et al., Proc. Natl. Acad. Sci. USA(1987) 84:7413-7416, which is herein incorporated by reference); mRNA(Malone et al., Proc. Natl. Acad. Sci. USA (1989) 86:6077-6081, which isherein incorporated by reference); and purified transcription factors(Debs et al., J. Biol. Chem. (1990) 265:10189-10192, which is hereinincorporated by reference), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areparticularly useful and are available under the trademark Lipofectin,from GIBCO BRL, Grand Island, N.Y. (See, also, Felgner et al., Proc.Natl. Acad. Sci. USA (1987) 84:7413-7416, which is herein incorporatedby reference). Other commercially available liposomes includetransfectace (DDAB/DOPE) and DOTAP/DOPE (Boehringer).

Other cationic liposomes can be prepared from readily availablematerials using techniques well known in the art. See, e.g. PCTPublication No. WO 90/11092 (which is herein incorporated by reference)for a description of the synthesis of DOTAP(1,2-bis(oleoyloxy)-3-(trimethylammonio)propane) liposomes. Preparationof DOTMA liposomes is explained in the literature, see, e.g., P. Felgneret al., Proc. Natl. Acad. Sci. USA 84:7413-7417, which is hereinincorporated by reference. Similar methods can be used to prepareliposomes from other cationic lipid materials.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidyl,choline, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

For example, commercially dioleoylphosphatidyl choline (DOPC),dioleoylphosphatidyl glycerol (DOPG), and dioleoylphosphatidylethanolamine (DOPE) can be used in various combinations to makeconventional liposomes, with or without the addition of cholesterol.Thus, for example, DOPG/DOPC vesicles can be prepared by drying 50 mgeach of DOPG and DOPC under a stream of nitrogen gas into a sonicationvial. The sample is placed under a vacuum pump overnight and is hydratedthe following day with deionized water. The sample is then sonicated for2 hours in a capped vial, using a Heat Systems model 350 sonicatorequipped with an inverted cup (bath type) probe at the maximum settingwhile the bath is circulated at 15EC. Alternatively, negatively chargedvesicles can be prepared without sonication to produce multilamellarvesicles or by extrusion through nucleopore membranes to produceunilamellar vesicles of discrete size. Other methods are known andavailable to those of skill in the art.

The liposomes can comprise multilamellar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs), withSUVs being preferred. The various liposome-nucleic acid complexes areprepared using methods well known in the art. See, e.g., Straubinger etal., Methods of Immunology (1983), 101:512-527, which is hereinincorporated by reference. For example, MLVs containing nucleic acid canbe prepared by depositing a thin film of phospholipid on the walls of aglass tube and subsequently hydrating with a solution of the material tobe encapsulated. SUVs are prepared by extended sonication of MLVs toproduce a homogeneous population of unilamellar liposomes. The materialto be entrapped is added to a suspension of preformed MLVs and thensonicated. When using liposomes containing cationic lipids, the driedlipid film is resuspended in an appropriate solution such as sterilewater or an isotonic buffer solution such as 10 mM Tris/NaCl, sonicated,and then the preformed liposomes are mixed directly with the DNA. Theliposome and DNA form a very stable complex due to binding of thepositively charged liposomes to the cationic DNA. SUVs find use withsmall nucleic acid fragments. LUVs are prepared by a number of methods,well known in the art. Commonly used methods include Ca²⁺-EDTA chelation(Papahadjopoulos et al., Biochim. Biophys. Acta (1975) 394:483; Wilsonet al., Cell (1979) 17:77); ether injection (Deamer, D. and Bangham, A.,Biochim. Biophys. Acta (1976) 443:629; Ostro et al., Biochem. Biophys.Res. Commun. (1977) 76:836; Fraley et al., Proc. Natl. Acad. Sci. USA(1979) 76:3348); detergent dialysis (Enoch, H. and Strittmatter, P.,Proc. Natl. Acad. Sci. USA (1979) 76:145); and reverse-phase evaporation(REV) (Fraley et al., J. Biol. Chem. (1980) 255:10431; Szoka, F. andPapahadjopoulos, D., Proc. Natl. Acad. Sci. USA (1978) 75:145;Schaefer-Ridder et al., Science (1982) 215:166), which are hereinincorporated by reference.

Generally, the ratio of DNA to liposomes will be from about 10:1 toabout 1:10. Preferably, the ration will be from about 5:1 to about 1:5.More preferably, the ration will be about 3:1 to about 1:3. Still morepreferably, the ratio will be about 1:1.

U.S. Pat. No. 5,676,954 (which is herein incorporated by reference)reports on the injection of genetic material, complexed with cationicliposomes carriers, into mice. U.S. Pat. Nos. 4,897,355, 4,946,787,5,049,386, 5,459,127, 5,589,466, 5,693,622, 5,580,859, 5,703,055, andinternational publication no. WO 94/9469 (which are herein incorporatedby reference) provide cationic lipids for use in transfecting DNA intocells and mammals. U.S. Pat. Nos. 5,589,466, 5,693,622, 5,580,859,5,703,055, and international publication no. WO 94/9469 (which areherein incorporated by reference) provide methods for deliveringDNA-cationic lipid complexes to mammals.

In certain embodiments, cells are be engineered, ex vivo or in vivo,using a retroviral particle containing RNA which comprises a sequenceencoding D-SLAM. Retroviruses from which the retroviral plasmid vectorsmay be derived include, but are not limited to, Moloney Murine LeukemiaVirus, spleen necrosis virus, Rous sarcoma Virus, Harvey Sarcoma Virus,avian leukosis virus, gibbon ape leukemia virus, human immunodeficiencyvirus, Myeloproliferative Sarcoma Virus, and mammary tumor virus.

The retroviral plasmid vector is employed to transduce packaging celllines to form producer cell lines. Examples of packaging cells which maybe transfected include, but are not limited to, the PE501, PA317, R-2,R-AM, PA12, T19-14X, VT-19-17-H2, RCRE, RCRIP, GP+E-86, GP+envAm12, andDAN cell lines as described in Miller, Human Gene Therapy 1:5-14 (1990),which is incorporated herein by reference in its entirety. The vectormay transduce the packaging cells through any means known in the art.Such means include, but are not limited to, electroporation, the use ofliposomes, and CaPO₄ precipitation. In one alternative, the retroviralplasmid vector may be encapsulated into a liposome, or coupled to alipid, and then administered to a host.

The producer cell line generates infectious retroviral vector particleswhich include polynucleotide encoding D-SLAM. Such retroviral vectorparticles then may be employed, to transduce eukaryotic cells, either invitro or in vivo. The transduced eukaryotic cells will express D-SLAM.

In certain other embodiments, cells are engineered, ex vivo or in vivo,with D-SLAM polynucleotide contained in an adenovirus vector. Adenoviruscan be manipulated such that it encodes and expresses D-SLAM, and at thesame time is inactivated in terms of its ability to replicate in anormal lytic viral life cycle. Adenovirus expression is achieved withoutintegration of the viral DNA into the host cell chromosome, therebyalleviating concerns about insertional mutagenesis. Furthermore,adenoviruses have been used as live enteric vaccines for many years withan excellent safety profile (Schwartz, A. R. et al. (1974) Am. Rev.Respir. Dis. 109:233-238). Finally, adenovirus mediated gene transferhas been demonstrated in a number of instances including transfer ofalpha-1-antitrypsin and CFTR to the lungs of cotton rats (Rosenfeld, M.A. et al. (1991) Science 252:431-434; Rosenfeld et al., (1992) Cell68:143-155). Furthermore, extensive studies to attempt to establishadenovirus as a causative agent in human cancer were uniformly negative(Green, M. et al. (1979) Proc. Natl. Acad. Sci. USA 76:6606).

Suitable adenoviral vectors useful in the present invention aredescribed, for example, in Kozarsky and Wilson, Curr. Opin. Genet.Devel. 3:499-503 (1993); Rosenfeld et al., Cell 68:143-155 (1992);Engelhardt et al., Human Genet. Ther. 4:759-769 (1993); Yang et al.,Nature Genet. 7:362-369 (1994); Wilson et al., Nature 365:691-692(1993); and U.S. Pat. No. 5,652,224, which are herein incorporated byreference. For example, the adenovirus vector Ad2 is useful and can begrown in human 293 cells. These cells contain the E1 region ofadenovirus and constitutively express E1a and E1b, which complement thedefective adenoviruses by providing the products of the genes deletedfrom the vector. In addition to Ad2, other varieties of adenovirus(e.g., Ad3, Ad5, and Ad7) are also useful in the present invention.

Preferably, the adenoviruses used in the present invention arereplication deficient. Replication deficient adenoviruses require theaid of a helper virus and/or packaging cell line to form infectiousparticles. The resulting virus is capable of infecting cells and canexpress a polynucleotide of interest which is operably linked to apromoter, for example, the HARP promoter of the present invention, butcannot replicate in most cells. Replication deficient adenoviruses maybe deleted in one or more of all or a portion of the following genes:E1a, E1b, E3, E4, E2a, or L1 through L5.

In certain other embodiments, the cells are engineered, ex vivo or invivo, using an adeno-associated virus (AAV). AAVs are naturallyoccurring defective viruses that require helper viruses to produceinfectious particles (Muzyczka, N., Curr. Topics in Microbiol. Immunol.158:97 (1992)). It is also one of the few viruses that may integrate itsDNA into non-dividing cells. Vectors containing as little as 300 basepairs of AAV can be packaged and can integrate, but space for exogenousDNA is limited to about 4.5 kb. Methods for producing and using suchAAVs are known in the art. See, for example, U.S. Pat. Nos. 5,139,941,5,173,414, 5,354,678, 5,436,146, 5,474,935, 5,478,745, and 5,589,377.

For example, an appropriate AAV vector for use in the present inventionwill include all the sequences necessary for DNA replication,encapsidation, and host-cell integration. The D-SLAM polynucleotideconstruct is inserted into the AAV vector using standard cloningmethods, such as those found in Sambrook et al., Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Press (1989). The recombinant AAVvector is then transfected into packaging cells which are infected witha helper virus, using any standard technique, including lipofection,electroporation, calcium phosphate precipitation, etc. Appropriatehelper viruses include adenoviruses, cytomegaloviruses, vacciniaviruses, or herpes viruses. Once the packaging cells are transfected andinfected, they will produce infectious AAV viral particles which containthe D-SLAM polynucleotide construct. These viral particles are then usedto transduce eukaryotic cells, either ex vivo or in vivo. The transducedcells will contain the D-SLAM polynucleotide construct integrated intoits genome, and will express D-SLAM.

Another method of gene therapy involves operably associatingheterologous control regions and endogenous polynucleotide sequences(e.g. encoding D-SLAM) via homologous recombination (see, e.g., U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not normally expressed in the cells, or isexpressed at a lower level than desired.

Polynucleotide constructs are made, using standard techniques known inthe art, which contain the promoter with targeting sequences flankingthe promoter. Suitable promoters are described herein. The targetingsequence is sufficiently complementary to an endogenous sequence topermit homologous recombination of the promoter-targeting sequence withthe endogenous sequence. The targeting sequence will be sufficientlynear the 5′ end of the D-SLAM desired endogenous polynucleotide sequenceso the promoter will be operably linked to the endogenous sequence uponhomologous recombination.

The promoter and the targeting sequences can be amplified using PCR.Preferably, the amplified promoter contains distinct restriction enzymesites on the 5′ and 3′ ends. Preferably, the 3′ end of the firsttargeting sequence contains the same restriction enzyme site as the 5′end of the amplified promoter and the 5′ end of the second targetingsequence contains the same restriction site as the 3′ end of theamplified promoter. The amplified promoter and targeting sequences aredigested and ligated together.

The promoter-targeting sequence construct is delivered to the cells,either as naked polynucleotide, or in conjunction withtransfection-facilitating agents, such as liposomes, viral sequences,viral particles, whole viruses, lipofection, precipitating agents, etc.,described in more detail above. The P promoter-targeting sequence can bedelivered by any method, included direct needle injection, intravenousinjection, topical administration, catheter infusion, particleaccelerators, etc. The methods are described in more detail below.

The promoter-targeting sequence construct is taken up by cells.Homologous recombination between the construct and the endogenoussequence takes place, such that an endogenous D-SLAM sequence is placedunder the control of the promoter. The promoter then drives theexpression of the endogenous D-SLAM sequence.

The polynucleotides encoding D-SLAM may be administered along with otherpolynucleotides encoding other angiogenic proteins. Angiogenic proteinsinclude, but are not limited to, acidic and basic fibroblast growthfactors, VEGF-1, epidermal growth factor alpha and beta,platelet-derived endothelial cell growth factor, platelet-derived growthfactor, tumor necrosis factor alpha, hepatocyte growth factor, insulinlike growth factor, colony stimulating factor, macrophage colonystimulating factor, granulocyte/macrophage colony stimulating factor,and nitric oxide synthase.

Preferably, the polynucleotide encoding D-SLAM contains a secretorysignal sequence that facilitates secretion of the protein. Typically,the signal sequence is positioned in the coding region of thepolynucleotide to be expressed towards or at the 5′ end of the codingregion. The signal sequence may be homologous or heterologous to thepolynucleotide of interest and may be homologous or heterologous to thecells to be transfected. Additionally, the signal sequence may bechemically synthesized using methods known in the art.

Any mode of administration of any of the above-described polynucleotidesconstructs can be used so long as the mode results in the expression ofone or more molecules in an amount sufficient to provide a therapeuticeffect. This includes direct needle injection, systemic injection,catheter infusion, biolistic injectors, particle accelerators (i.e.,“gene guns”), gelfoam sponge depots, other commercially available depotmaterials, osmotic pumps (e.g., Alza minipumps), oral or suppositorialsolid (tablet or pill) pharmaceutical formulations, and decanting ortopical applications during surgery. For example, direct injection ofnaked calcium phosphate-precipitated plasmid into rat liver and ratspleen or a protein-coated plasmid into the portal vein has resulted ingene expression of the foreign gene in the rat livers (Kaneda et al.,Science 243:375 (1989)).

A preferred method of local administration is by direct injection.Preferably, a recombinant molecule of the present invention complexedwith a delivery vehicle is administered by direct injection into orlocally within the area of arteries. Administration of a compositionlocally within the area of arteries refers to injecting the compositioncentimeters and preferably, millimeters within arteries.

Another method of local administration is to contact a polynucleotideconstruct of the present invention in or around a surgical wound. Forexample, a patient can undergo surgery and the polynucleotide constructcan be coated on the surface of tissue inside the wound or the constructcan be injected into areas of tissue inside the wound.

Therapeutic compositions useful in systemic administration, includerecombinant molecules of the present invention complexed to a targeteddelivery vehicle of the present invention. Suitable delivery vehiclesfor use with systemic administration comprise liposomes comprisingligands for targeting the vehicle to a particular site.

Preferred methods of systemic administration, include intravenousinjection, aerosol, oral and percutaneous (topical) delivery.Intravenous injections can be performed using methods standard in theart. Aerosol delivery can also be performed using methods standard inthe art (see, for example, Stribling et al., Proc. Natl. Acad. Sci. USA189:11277-11281, 1992, which is incorporated herein by reference). Oraldelivery can be performed by complexing a polynucleotide construct ofthe present invention to a carrier capable of withstanding degradationby digestive enzymes in the gut of an animal. Examples of such carriers,include plastic capsules or tablets, such as those known in the art.Topical delivery can be performed by mixing a polynucleotide constructof the present invention with a lipophilic reagent (e.g., DMSO) that iscapable of passing into the skin.

Determining an effective amount of substance to be delivered can dependupon a number of factors including, for example, the chemical structureand biological activity of the substance, the age and weight of theanimal, the precise condition requiring treatment and its severity, andthe route of administration. The frequency of treatments depends upon anumber of factors, such as the amount of polynucleotide constructsadministered per dose, as well as the health and history of the subject.The precise amount, number of doses, and timing of doses will bedetermined by the attending physician or veterinarian.

Therapeutic compositions of the present invention can be administered toany animal, preferably to mammals and birds. Preferred mammals includehumans, dogs, cats, mice, rats, rabbits sheep, cattle, horses and pigs,with humans being particularly preferred.

Biological Activities of D-SLAM

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, can be used in assays to test for one or more biologicalactivities. If D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, do exhibit activity in a particular assay, it islikely that D-SLAM may be involved in the diseases associated with thebiological activity. Therefore, D-SLAM could be used to treat, diagnose,detect, and/or prevent the associated disease.

D-SLAM is a cell surface receptor homologous to members of the SecretedLymphocyte Activation Molecule (SLAM) family, and thus should haveactivity similar to other SLAM family members. Current studies in theliterature demonstrate that SLAM can associate with itself, and thatthis homotypic interaction can activate B- and T-cells. Therefore,D-SLAM may interact specifically with SLAM, with D-SLAM (a homotypicinteraction), or other B- and T-cell receptor molecules on the surfaceof B- and T-cells to affect the activation, proliferation, survival,and/or differentiation of immune cells. Similarly, soluble D-SLAM may bean important costimulatory molecule for therapeutic uses or immunemodulation. Ligands, such as antibodies, may mimic the action of solubleD-SLAM by binding to D-SLAM, SLAM, or other dendritic cell receptors.

D-SLAM inhibits the proliferation of B-cells. See, the experimentalresults shown in Examples 32-33, below.

In a preferred embodiment of the invention, D-SLAM and/or D-SLAMagonists can be used to inhibit the proliferation of B-cells. Thus,D-SLAM and/or D-SLAM agonists would be useful in treating, preventing,diagnosing, detecting, and/or ameliorating diseases and/or disordersrelated to an overproliferation of B-cells.

In a preferred embodiment of the invention, D-SLAM antagonists can beused to promote the proliferation of B-cells. Thus, D-SLAM antagonistswould be useful in treating, preventing, diagnosing, detecting, and/orameliorating diseases and/or disorders related to a reducedproliferation of B-cells.

Binding of D-SLAM induces the production of interferon-gamma from othercell types, particularly T- and B-cells (data not shown.) The bindingmay occur through homotypic association with membrane bound D-SLAM,association with SLAM, or association with other T- or B-cell receptors.Ligands, such as antibodies, may mimic the induction of interferon-gammaby soluble D-SLAM by binding to D-SLAM, SLAM, or other dendritic cellreceptors.

Moreover, because of the tissue distribution of D-SLAM, this protein mayalso play a role in stimulating dendritic or antigen presenting cells.For example, a secreted form of D-SLAM, containing the extracellulardomain or the full-length form, may bind to and stimulate D-SLAMmolecules located on the surface of dendritic or antigen-presentingcells in homotypic manner. Binding may also occur to SLAM, or otherdendritic cell surface receptors. This binding may regulate thesurvival, proliferation, differentiation, activation or maturation ofdendritic cells or antigen presenting cells, effecting antigenrecognition and immune response. Moreover, ligands, such as antibodies,may mimic the action of soluble D-SLAM by binding to D-SLAM, SLAM, orother dendritic cell receptors.

Thus, D-SLAM may be useful as a therapeutic molecule. It could be usedto control the proliferation, activation, maturation, survival, and/ordifferentiation of hematopoietic cells, in particular B- and T-cells.Particularly, D-SLAM may be a useful therapeutic to mediate immunemodulation, and may influence the Th0-TH1-TH2 profile of a patient'simmune system. For example, D-SLAM may drive immune response to theTh0-TH1 pathway. This control of immune cells would be particularlyimportant in the treatment, diagnosis, detection, and/or prevention ofimmune disorders, such as autoimmune diseases or immunosuppression (seebelow). Preferably, treatment, diagnosis, detection, and/or preventionof immune disorders could be carried out using a secreted form ofD-SLAM, gene therapy, or ex vivo applications. Moreover, inhibitors ofD-SLAM, either blocking antibodies or mutant forms, could modulate theexpression of D-SLAM. These inhibitors may be useful to treat, diagnose,detect, and/or prevent diseases associated with the misregulation ofD-SLAM, such as T cell lymphoma.

In one embodiment, the invention provides a method for the specificdelivery of compositions of the invention to cells by administeringpolypeptides of the invention (e.g., D-SLAM polypeptides or anti-D-SLAMantibodies) that are associated with heterologous polypeptides ornucleic acids. In one example, the invention provides a method fordelivering a therapeutic protein into the targeted cell. In anotherexample, the invention provides a method for delivering a singlestranded nucleic acid (e.g., antisense or ribozymes) or double strandednucleic acid (e.g., DNA that can integrate into the cell's genome orreplicate episomally and that can be transcribed) into the targetedcell.

In another embodiment, the invention provides a method for the specificdestruction of cells (e.g., the destruction of tumor cells) byadministering polypeptides of the invention (e.g., D-SLAM polypeptidesor anti-D-SLAM antibodies) in association with toxins or cytotoxicprodrugs.

By “toxin” is meant compounds that bind and activate endogenouscytotoxic effector systems, radioisotopes, holotoxins, modified toxins,catalytic subunits of toxins, cytotoxins (cytotoxic agents), or anymolecules or enzymes not normally present in or on the surface of a cellthat under defined conditions cause the cell's death. Toxins that may beused according to the methods of the invention include, but are notlimited to, radioisotopes known in the art, compounds such as, forexample, antibodies (or complement fixing containing portions thereof)that bind an inherent or induced endogenous cytotoxic effector system,thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin,Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin,pokeweed antiviral protein, alpha-sarcin and cholera toxin. “Toxin” alsoincludes a cytostatic or cytocidal agent, a therapeutic agent or aradioactive metal ion, e.g., alpha-emitters such as, for example, ²¹³Bi,or other radioisotopes such as, for example, ¹⁰³Pd, ¹³³Xe, ¹³¹I, ⁶⁸Ge,⁵⁷Co, ⁶⁵Zn, ⁸⁵Sr, ³²P, ³⁵S, ⁹⁰Y, ¹⁵³Sm, ¹⁵³Gd, ¹⁶⁹Yb, ⁵¹Cr, ⁵⁴Mn, ⁷⁵Se,¹¹³Sn, ⁹⁰Yttrium, ¹¹⁷Tin, ¹⁸⁶Rhenium, ¹⁶⁶Holmium, and ¹⁸⁸Rhenium;luminescent labels, such as luminol; and fluorescent labels, such asfluorescein and rhodamine, and biotin.

Techniques known in the art may be applied to label antibodies of theinvention. Such techniques include, but are not limited to, the use ofbifunctional conjugating agents (see e.g., U.S. Pat. Nos. 5,756,065;5,714,631; 5,696,239; 5,652,361; 5,505,931; 5,489,425; 5,435,990;5,428,139; 5,342,604; 5,274,119; 4,994,560; and 5,808,003; the contentsof each of which are hereby incorporated by reference in its entirety).A cytotoxin or cytotoxic agent includes any agent that is detrimental tocells. Examples include paclitaxol, cytochalasin B, gramicidin D,ethidium bromide, emetine, mitomycin, etoposide, tenoposide,vincristine, vinblastine, colchicin, doxorubicin, daunorubicin,dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine,propranolol, and puromycin and analogs or homologs thereof. Therapeuticagents include, but are not limited to, antimetabolites (e.g.,methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine,5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine,thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU),cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycinC, and cis-dichlorodiamine platinum (II) (DDP) cisplatin),anthracyclines (e.g., daunorubicin (formerly daunomycin) anddoxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin),bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents(e.g., vincristine and vinblastine).

By “cytotoxic prodrug” is meant a non-toxic compound that is convertedby an enzyme, normally present in the cell, into a cytotoxic compound.Cytotoxic prodrugs that may be used according to the methods of theinvention include, but are not limited to, glutamyl derivatives ofbenzoic acid mustard alkylating agent, phosphate derivatives ofetoposide or mitomycin C, cytosine arabinoside, daunorubisin, andphenoxyacetamide derivatives of doxorubicin.

It will be appreciated that conditions caused by a decrease in thestandard or normal level of D-SLAM activity in an individual,particularly disorders of the immune system, can be treated byadministration of D-SLAM polypeptide (in the form of solubleextracellular domain or cells expressing the complete protein) oragonist. Thus, the invention also provides a method of treatment of anindividual in need of an increased level of D-SLAM activity comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of an isolated D-SLAM polypeptide of the invention,or agonist thereof (e.g, an agonistic D-SLAM antibody), effective toincrease the D-SLAM activity level in such an individual.

It will also be appreciated that conditions caused by a increase in thestandard or normal level of D-SLAM activity in an individual,particularly disorders of the immune system, can be treated byadministration of D-SLAM polypeptides (in the form of solubleextracellular domain or cells expressing the complete protein) orantagonist (e.g, an antagonistic D-SLAM antibody). Thus, the inventionalso provides a method of treatment of an individual in need of andecreased level of D-SLAM activity comprising administering to such anindividual a pharmaceutical composition comprising an amount of anisolated D-SLAM polypeptide of the invention, or antagonist thereof,effective to decrease the D-SLAM activity level in such an individual.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated by D-SLAM polynucleotides or polypeptides,or agonists of D-SLAM. Examples of viruses, include, but are not limitedto the following DNA and RNA viruses and viral families: Arbovirus,Adenoviridae, Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae,Caliciviridae, Circoviridae, Coronaviridae, Dengue, EBV, HIV,Flaviviridae, Hepadnaviridae (Hepatitis), Herpesviridae (such as,Cytomegalovirus, Herpes Simplex, Herpes Zoster), Mononegavirus (e.g.,Paramyxoviridae, Morbillivirus, Rhabdoviridae), Orthomyxoviridae (e.g.,Influenza A, Influenza B, and parainfluenza), Papiloma virus,Papovaviridae, Parvoviridae, Picomaviridae, Poxyiridae (such as Smallpoxor Vaccinia), Reoviridae (e.g., Rotavirus), Retroviridae (HTLV-I,HTLV-II, Lentivirus), and Togaviridae (e.g., Rubivirus). Viruses fallingwithin these families can cause a variety of diseases or symptoms,including, but not limited to: arthritis, bronchiollitis, respiratorysyncytial virus, encephalitis, eye infections (e.g., conjunctivitis,keratitis), chronic fatigue syndrome, hepatitis (A, B, C, E, ChronicActive, Delta), Japanese B encephalitis, Junin, Chikungunya, Rift Valleyfever, yellow fever, meningitis, opportunistic infections (e.g., AIDS),pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagic fever, Measles,Mumps, Parainfluenza, Rabies, the common cold, Polio, leukemia, Rubella,sexually transmitted diseases, skin diseases (e.g., Kaposi's, warts),and viremia. D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can be used to treat, prevent, diagnose, and/ordetect any of these symptoms or diseases. In specific embodiments,D-SLAM polynucleotides, polypeptides, or agonists are used to treat,prevent, and/or diagnose: meningitis, Dengue, EBV, and/or hepatitis(e.g., hepatitis B). In an additional specific embodiment D-SLAMpolynucleotides, polypeptides, or agonists are used to treat patientsnonresponsive to one or more other commercially available hepatitisvaccines. In a further specific embodiment, D-SLAM polynucleotides,polypeptides, or agonists are used to treat, prevent, and/or diagnoseAIDS. In an additional specific embodiment D-SLAM polynucleotides,polypeptides, agonists, and/or antagonists are used to treat, prevent,and/or diagnose patients with cryptosporidiosis.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated by D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, include, but not limited to, thefollowing Gram-Negative and Gram-positive bacteria and bacterialfamilies and fungi: Actinomycetales (e.g., Corynebacterium,Mycobacterium, Norcardia), Cryptococcus neoformans, Aspergillosis,Bacillaceae (e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis,Bordetella, Borrelia (e.g., Borrelia burgdorferi, Brucellosis,Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis,Dermatocycoses, E. coli (e.g., Enterotoxigenic E. coli andEnterohemorrhagic E. coli), Enterobacteriaceae (Klebsiella, Salmonella(e.g., Salmonella typhi, and Salmonella paratyphi), Serratia, Yersinia),Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria(e.g, Listeria monocytogenes), Mycoplasmatales, Mycobacterium leprae,Vibrio cholerae, Neisseriaceae (e.g., Acinetobacter, Gonorrhea,Menigococcal), Meisseria meningitidis, Pasteurellacea Infections (e.g.,Actinobacillus, Heamophilus (e.g., Heamophilus influenza type B),Pasteurella), Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis,Shigella spp., Staphylococcal, Meningiococcal, Pneumococcal andStreptococcal (e.g., Streptococcus pneumoniae and Group BStreptococcus). These bacterial or fungal families can cause thefollowing diseases or symptoms, including, but not limited to:bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis,uveitis), gingivitis, opportunistic infections (e.g., AIDS relatedinfections), paronychia, prosthesis-related infections, Reiter'sDisease, respiratory tract infections, such as Whooping Cough orEmpyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery,Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea,meningitis (e.g., mengitis types A and B), Chlamydia, Syphilis,Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism,gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexuallytransmitted diseases, skin diseases (e.g., cellulitis, dermatocycoses),toxemia, urinary tract infections, wound infections. D-SLAMpolynucleotides or polypeptides, or agonists or antagonists of D-SLAM,can be used to treat, prevent, diagnose, and/or detect any of thesesymptoms or diseases. In specific embodiments, D-SLAM polynucleotides,polypeptides, or antagonists thereof are used to treat, prevent, and/ordiagnose: tetanus, Diptheria, botulism, and/or meningitis type B.

Moreover, parasitic agents causing disease or symptoms that can betreated by D-SLAM polynucleotides or polypeptides, or antagonists ofD-SLAM, include, but not limited to, the following families or class:Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis,Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis,Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas andSporozoans (e.g., Plasmodium virax, Plasmodium falciparium, Plasmodiummalariae and Plasmodium ovale). These parasites can cause a variety ofdiseases or symptoms, including, but not limited to: Scabies,Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,giardiasis), liver disease, lung disease, opportunistic infections(e.g., AIDS related), malaria, pregnancy complications, andtoxoplasmosis. D-SLAM polynucleotides or polypeptides, or antagonists orantagonists of D-SLAM, can be used to treat, prevent, diagnose, and/ordetect any of these symptoms or diseases. In specific embodiments,D-SLAM polynucleotides, polypeptides, or antagonists thereof are used totreat, prevent, and/or diagnose malaria.

In another embodiment, D-SLAM polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, are used to treat,prevent, and/or diagnose inner ear infection (such as, for example,otitis media), as well as other infections characterized by infectionwith Streptococcus pneumoniae and other pathogenic organisms.

In a specific embodiment, D-SLAM polynucleotides or polypeptides, oragonists or antagonists thereof (e.g., anti-D-SLAM antibodies) are usedto treat or prevent a disorder characterized by deficient serumimmunoglobulin production, recurrent infections, and/or immune systemdysfunction. Moreover, D-SLAM polynucleotides or polypeptides, oragonists or antagonists thereof (e.g., anti-D-SLAM antibodies) may beused to treat or prevent infections of the joints, bones, skin, and/orparotid glands, blood-borne infections (e.g., sepsis, meningitis, septicarthritis, and/or osteomyelitis), autoimmune diseases (e.g., thosedisclosed herein), inflammatory disorders, and malignancies, and/or anydisease or disorder or condition associated with these infections,diseases, disorders and/or malignancies) including, but not limited to,CVID, other primary immune deficiencies, HIV disease, CLL, recurrentbronchitis, sinusitis, otitis media, conjunctivitis, pneumonia,hepatitis, meningitis, herpes zoster (e.g., severe herpes zoster),and/or pheumocystis carnii.

D-SLAM polynucleotides or polypeptides of the invention, or agonists orantagonists thereof, may be used to diagnose, prognose, treat or preventone or more of the following diseases or disorders, or conditionsassociated therewith: primary immuodeficiencies, immune-mediatedthrombocytopenia, Kawasaki syndrome, bone marrow transplant (e.g.,recent bone marrow transplant in adults or children), chronic B-celllymphocytic leukemia, HIV infection (e.g., adult or pediatric HIVinfection), chronic inflammatory demyelinating polyneuropathy, andpost-transfusion purpura.

Additionally, D-SLAM polynucleotides or polypeptides of the invention,or agonists or antagonists thereof, may be used to diagnose, prognose,treat or prevent one or more of the following diseases, disorders, orconditions associated therewith, Guillain-Barre syndrome, anemia (e.g.,anemia associated with parvovirus B19, patients with stable mutliplemyeloma who are at high risk for infection (e.g., recurrent infection),autoimmune hemolytic anemia (e.g., warm-type autoimmune hemolyticanemia), thrombocytopenia (e.g, neonatal thrombocytopenia), andimmune-mediated neutropenia), transplantation (e.g, cytamegalovirus(CMV)-negative recipients of CMV-positive organs), hypogammaglobulinemia(e.g., hypogammaglobulinemic neonates with risk factor for infection ormorbidity), epilepsy (e.g, intractable epilepsy), systemic vasculiticsyndromes, myasthenia gravis (e.g, decompensation in myasthenia gravis),dermatomyositis, and polymyositis.

Additional preferred embodiments of the invention include, but are notlimited to, the use of D-SLAM polypeptides, D-SLAM polynucleotides, andfunctional agonists and/or antagonists thereof, in the followingapplications:

Administration to an animal (e.g., mouse, rat, rabbit, hamster, guineapig, pigs, micro-pig, chicken, camel, goat, horse, cow, sheep, dog, cat,non-human primate, and human, most preferably human) to inhibit theimmune system to produce decreased quantities of one or more antibodies(e.g., IgG, IgA, IgM, and IgE), to inhibit higher affinity antibodyproduction (e.g., IgG, IgA, IgM, and IgE), and/or to decrease an immuneresponse. In a specific nonexclusive embodiment, D-SLAM polypeptides ofthe invention, and/or agonists thereof, are administered to inhibit theimmune system to produce decreased quantities of IgG. In anotherspecific nonexclusive embodiment, D-SLAM polypeptides of the inventionand/or agonists thereof, are administered to boost the immune system toproduce decreased quantities of IgA. In another specific nonexclusiveembodiment, D-SLAM polypeptides of the invention and/or agoniststhereof, are administered to inhibit the immune system to producedecreased quantities of IgM.

As an agent that reduces the immune status of an individual prior totheir receipt of immunosuppressive therapies.

As an agent to decrease serum immunoglobulin concentrations.

As an immune system inhibitor prior to, during, or after bone marrowtransplant and/or other transplants (e.g., allogeneic or xenogeneicorgan transplantation). With respect to transplantation, compositions ofthe invention may be administered prior to, concomitant with, and/orafter transplantation.

As an agent to reduce immunoresponsiveness. B cell immunodeficienciesthat may be ameliorated or treated by administering D-SLAM antagonistsof the invention include, but are not limited to, severe combinedimmunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminasedeficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton'sdisease, congenital agammaglobulinemia, X-linked infantileagammaglobulinemia, acquired agammaglobulinemia, adult onsetagammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia,hypogammaglobulinemia, transient hypogammaglobulinemia of infancy,unspecified hypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymphoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

D-SLAM antagonists may be used as agents to boost immunoresponsivenessamong individuals having an acquired loss of B cell function. Conditionsresulting in an acquired loss of B cell function that may be amelioratedor treated by administering the D-SLAM antagonists of the inventioninclude, but are not limited to, HIV Infection, AIDS, bone marrowtransplant, and B cell chronic lymphocytic leukemia (CLL).

D-SLAM antagonists may be used as agents to boost immunoresponsivenessamong individuals having a temporary immune deficiency. Conditionsresulting in a temporary immune deficiency that may be ameliorated ortreated by administering D-SLAM antagonists include, but are not limitedto, recovery from viral infections (e.g., influenza), conditionsassociated with malnutrition, recovery from infectious mononucleosis, orconditions associated with stress, recovery from measles, recovery fromblood transfusion, recovery from surgery.

As an agent to direct an individual's immune system towards developmentof a humoral response (i.e. TH2) as opposed to a TH1 cellular response.

As a means to inhibit tumor proliferation.

As a therapy for generation and/or regeneration of lymphoid tissuesfollowing surgery, trauma or genetic defect.

As a gene-based therapy for genetically inherited disorders resulting inimmuno-incompetence such as observed among SCID patients.

As an antigen for the generation of antibodies to inhibit or enhanceD-SLAM-mediated responses.

As a means of inhibiting monocytes/macrophages to defend againstparasitic diseases that effect monocytes such as Leshmania.

As pretreatment of bone marrow samples prior to transplant. Suchtreatment would decrease B cell representation and thus modulaterecovery.

As a means of regulating secreted cytokines that are elicited by D-SLAM.

D-SLAM polypeptides or polynucleotides of the invention, or antagonistsmay be used to modulate IgE concentrations in vitro or in vivo.

Additionally, D-SLAM polypeptides or polynucleotides of the invention,or antagonists thereof, may be used to treat, prevent, and/or diagnoseIgE-mediated allergic reactions. Such allergic reactions include, butare not limited to, asthma, rhinitis, and eczema.

In a specific embodiment, D-SLAM polypeptides or polynucleotides of theinvention, or antagonists thereof, is administered to treat, prevent,diagnose, and/or ameliorate selective IgA deficiency.

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate ataxia-telangiectasia.

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate common variable immunodeficiency.

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate X-linked agammaglobulinemia.

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate severe combined immunodeficiency(SCID).

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate Wiskott-Aldrich syndrome.

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate X-linked Ig deficiency with hyperIgM.

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists or antagonists (e.g., anti-D-SLAMantibodies) thereof, is administered to treat, prevent, and/or diagnosechronic myelogenous leukemia, acute myelogenous leukemia, leukemia,hystiocytic leukemia, monocytic leukemia (e.g., acute monocyticleukemia), leukemic reticulosis, Shilling Type monocytic leukemia,and/or other leukemias derived from monocytes and/or monocytic cellsand/or tissues.

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate monocytic leukemoid reaction, asseen, for example, with tuberculosis.

In another specific embodiment, D-SLAM polypeptides or polynucleotidesof the invention, or antagonists thereof, is administered to treat,prevent, diagnose, and/or ameliorate monocytic leukocytosis, monocyticleukopenia, monocytopenia, and/or monocytosis.

In a specific embodiment, D-SLAM polynucleotides or polypeptides of theinvention, and/or anti-D-SLAM antibodies and/or agonists or antagoniststhereof, are used to treat, prevent, detect, and/or diagnose primary Blymphocyte disorders and/or diseases, and/or conditions associatedtherewith.

In a preferred embodiment, D-SLAM polynucleotides, polypeptides, and/oragonists and/or antagonists thereof are used to treat, prevent, and/ordiagnose diseases or disorders affecting or conditions associated withany one or more of the various mucous membranes of the body. Suchdiseases or disorders include, but are not limited to, for example,mucositis, mucoclasis, mucocolitis, mucocutaneous leishmaniasis (suchas, for example, American leishmaniasis, leishmaniasis americana,nasopharyngeal leishmaniasis, and New World leishmaniasis),mucocutaneous lymph node syndrome (for example, Kawasaki disease),mucoenteritis, mucoepidermoid carcinoma, mucoepidermoid tumor,mucoepithelial dysplasia, mucoid adenocarcinoma, mucoid degeneration,myxoid degeneration; myxomatous degeneration; myxomatosis, mucoid medialdegeneration (for example, cystic medial necrosis), mucolipidosis(including, for example, mucolipidosis I, mucolipidosis II,mucolipidosis m, and mucolipidosis IV), mucolysis disorders,mucomembranous enteritis, mucoenteritis, mucopolysaccharidosis (such as,for example, type I mucopolysaccharidosis (i.e., Hurler's syndrome),type IS mucopolysaccharidosis (i.e., Scheie's syndrome or type Vmucopolysaccharidosis), type II mucopolysaccharidosis (i.e., Hunter'ssyndrome), type III mucopolysaccharidosis (i.e., Sanfilippo's syndrome),type IV mucopolysaccharidosis (i.e., Morquio's syndrome), type VImucopolysaccharidosis (i.e., Maroteaux-Lamy syndrome), type VIImucopolysaccharidosis (i.e, mucopolysaccharidosis due tobeta-glucuronidase deficiency), and mucosulfatidosis),mucopolysacchariduria, mucopurulent conjunctivitis, mucopus,mucormycosis (i.e., zygomycosis), mucosal disease (i.e., bovine virusdiarrhea), mucous colitis (such as, for example, mucocolitis andmyxomembranous colitis), and mucoviscidosis (such as, for example,cystic fibrosis, cystic fibrosis of the pancreas, Clarke-Hadfieldsyndrome, fibrocystic disease of the pancreas, mucoviscidosis, andviscidosis). In a highly preferred embodiment, D-SLAM polynucleotides,polypeptides, and/or agonists and/or antagonists thereof are used totreat, prevent, and/or diagnose mucositis, especially as associated withchemotherapy.

In a preferred embodiment, D-SLAM polynucleotides, polypeptides, and/oragonists and/or antagonists thereof are used to treat, prevent, and/ordiagnose diseases or disorders affecting or conditions associated withsinusitis.

An additional condition, disease or symptom that can be treated,prevented, and/or diagnosed by D-SLAM polynucleotides or polypeptides,or antagonists of D-SLAM, is osteomyelitis.

An additional condition, disease or symptom that can be treated,prevented, and/or diagnosed by D-SLAM polynucleotides or polypeptides,or antagonists of D-SLAM, is endocarditis.

All of the above described applications as they may apply to veterinarymedicine.

J D-SLAM antagonists may be used as a therapy for B cell malignanciessuch as ALL, Hodgkins disease, non-Hodgkins lymphoma, Chronic lymphocyteleukemia, plasmacytomas, multiple myeloma, Burkitt's lymphoma, andEBV-transformed diseases, as well as a therapy for chronichypergammaglobulinemeia evident in such diseases as monoclonalgammopathyof undetermined significance (MGUS), Waldenstrom's disease, relatedidiopathic monoclonalgammopathies, and plasmacytomas.

An immunosuppressive agent(s).

D-SLAM polypeptides or polynucleotides of the invention, or antagonistsmay be used to modulate IgE concentrations in vitro or in vivo.

In another embodiment, administration of D-SLAM polypeptides orpolynucleotides of the invention, or antagonists thereof, may be used totreat, prevent, and/or diagnose IgE-mediated allergic reactionsincluding, but not limited to, asthma, rhinitis, and eczema.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

The agonists and antagonists may be employed in a composition with apharmaceutically acceptable carrier, e.g., as described herein.

All of the above described applications as they may apply to veterinarymedicine. Moreover, all applications described herein may also apply toveterinary medicine.

D-SLAM polynucleotides or polypeptides of the invention and/or agonistsand/or antagonists thereof, may be used to treat, prevent, and/ordiagnose various immune system-related disorders and/or conditionsassociated with these disorders, in mammals, preferably humans. Manyautoimmune disorders result from inappropriate recognition of self asforeign material by immune cells. This inappropriate recognition resultsin an immune response leading to the destruction of the host tissue.Therefore, the administration of D-SLAM polynucleotides or polypeptidesof the invention and/or agonists and/or agonists thereof that caninhibit an immune response, particularly the proliferation of B cellsand/or the production of immunoglobulins, may be an effective therapy intreating and/or preventing autoimmune disorders. Thus, in preferredembodiments, D-SLAM agonists of the invention are used to treat,prevent, and/or diagnose an autoimmune disorder.

Autoimmune disorders and conditions associated with these disorders thatmay be treated, prevented, and/or diagnosed with the D-SLAMpolynucleotides, polypeptides, and/or agonists of the invention,include, but are not limited to, autoimmune hemolytic anemia, autoimmuneneonatal thrombocytopenia, idiopathic thrombocytopenia purpura,autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome,dermatitis, allergic encephalomyelitis, myocarditis, relapsingpolychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgAnephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia,Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpura),Reiter's Disease, Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation,Guillain-Barre Syndrome, insulin dependent diabetes mellitis, andautoimmune inflammatory eye disease.

Additional autoimmune disorders (that are highly probable) that may betreated, prevented, and/or diagnosed with the compositions of theinvention include, but are not limited to, autoimmune thyroiditis,hypothyroidism (i.e., Hashimoto's thyroiditis) (often characterized,e.g., by cell-mediated and humoral thyroid cytotoxicity), systemic lupuserhythematosus (often characterized, e.g., by circulating and locallygenerated immune complexes), Goodpasture's syndrome (oftencharacterized, e.g., by anti-basement membrane antibodies), Pemphigus(often characterized, e.g., by epidermal acantholytic antibodies),Receptor autoimmunities such as, for example, (a) Graves' Disease (oftencharacterized, e.g., by TSH receptor antibodies), (b) Myasthenia Gravis(often characterized, e.g., by acetylcholine receptor antibodies), and(c) insulin resistance (often characterized, e.g., by insulin receptorantibodies), autoimmune hemolytic anemia (often characterized, e.g., byphagocytosis of antibody-sensitized RBCs), autoimmune thrombocytopenicpurpura (often characterized, e.g., by phagocytosis ofantibody-sensitized platelets.

Additional autoimmune disorders (that are probable) that may be treated,prevented, and/or diagnosed with the compositions of the inventioninclude, but are not limited to, rheumatoid arthritis (oftencharacterized, e.g., by immune complexes in joints), schleroderma withanti-collagen antibodies (often characterized, e.g., by nucleolar andother nuclear antibodies), mixed connective tissue disease (oftencharacterized, e.g., by antibodies to extractable nuclear antigens(e.g., ribonucleoprotein)), polymyositis/dermatomyositis (oftencharacterized, e.g., by nonhistone ANA), pernicious anemia (oftencharacterized, e.g., by antiparietal cell, microsomes, and intrinsicfactor antibodies), idiopathic Addison's disease (often characterized,e.g., by humoral and cell-mediated adrenal cytotoxicity, infertility(often characterized, e.g., by antispermatozoal antibodies),glomerulonephritis (often characterized, e.g., by glomerular basementmembrane antibodies or immune complexes) such as primaryglomerulonephritis and IgA nephropathy, bullous pemphigoid (oftencharacterized, e.g., by IgG and complement in basement membrane),Sjogren's syndrome (often characterized, e.g., by multiple tissueantibodies, and/or a specific nonhistone ANA (SS-B)), diabetes millitus(often characterized, e.g., by cell-mediated and humoral islet cellantibodies), and adrenergic drug resistance (including adrenergic drugresistance with asthma or cystic fibrosis) (often characterized, e.g.,by beta-adrenergic receptor antibodies).

Additional autoimmune disorders (that are possible) that may be treated,prevented, and/or diagnosed with the compositions of the inventioninclude, but are not limited to, chronic active hepatitis (oftencharacterized, e.g., by smooth muscle antibodies), primary biliarycirrhosis (often characterized, e.g., by mitchondrial antibodies), otherendocrine gland failure (often characterized, e.g., by specific tissueantibodies in some cases), vitiligo (often characterized, e.g., bymelanocyte antibodies), vasculitis (often characterized, e.g., by Ig andcomplement in vessel walls and/or low serum complement), post-MI (oftencharacterized, e.g., by myocardial antibodies), cardiotomy syndrome(often characterized, e.g., by myocardial antibodies), urticaria (oftencharacterized, e.g., by IgG and IgM antibodies to IgE), atopicdermatitis (often characterized, e.g., by IgG and IgM antibodies toIgE), asthma (often characterized, e.g., by IgG and IgM antibodies toIgE), inflammatory myopathies, and many other inflammatory,granulamatous, degenerative, and atrophic disorders.

In a preferred embodiment, the autoimmune diseases and disorders and/orconditions associated with the diseases and disorders recited above aretreated, prevented, and/or diagnosed using anti-D-SLAM antibodies.

In a specific preferred embodiment, rheumatoid arthritis is treated,prevented, and/or diagnosed using D-SLAM and/or other agonists of theinvention.

In a specific preferred embodiment, lupus is treated, prevented, and/ordiagnosed using D-SLAM and/or other agonists of the invention.

In a specific preferred embodiment, nephritis associated with lupus istreated, prevented, and/or diagnosed using D-SLAM and/or other agonistsof the invention.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated by D-SLAM polynucleotides or polypeptides of the inventionand/or agonists and/or antagonists thereof. Moreover, these moleculescan be used to treat, prevent, and/or diagnose anaphylaxis,hypersensitivity to an antigenic molecule, or blood groupincompatibility.

D-SLAM polynucleotides or polypeptides of the invention and/or agonistsand/or antagonists thereof, may also be used to treat, prevent, and/ordiagnose organ rejection or graft-versus-host disease (GVHD) and/orconditions associated therewith. Organ rejection occurs by host immunecell destruction of the transplanted tissue through an immune response.Similarly, an immune response is also involved in GVHD, but, in thiscase, the foreign transplanted immune cells destroy the host tissues.The administration of D-SLAM polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, that inhibits animmune response, particularly the proliferation, differentiation, orchemotaxis of T-cells, may be an effective therapy in preventing organrejection or GVHD.

Similarly, D-SLAM polynucleotides or polypeptides of the inventionand/or agonists and/or antagonists thereof, may also be used to modulateinflammation. For example, D-SLAM polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, may inhibit theproliferation and differentiation of cells involved in an inflammatoryresponse. These molecules can be used to treat, prevent, and/or diagnoseinflammatory conditions, both chronic and acute conditions, includingchronic prostatitis, granulomatous prostatitis and malacoplakia,inflammation associated with infection (e.g., septic shock, sepsis, orsystemic inflammatory response syndrome (SIRS)), ischemia-reperfusioninjury, endotoxin lethality, arthritis, complement-mediated hyperacuterejection, nephritis, cytokine or chemokine induced lung injury,inflammatory bowel disease, Crohn's disease, or resulting from overproduction of cytokines (e.g., TNF or IL-1.)

In a specific embodiment, anti-D-SLAM antibodies of the invention areused to treat, prevent, modulate, detect, and/or diagnose inflammation.

In a specific embodiment, anti-D-SLAM antibodies of the invention areused to treat, prevent, modulate, detect, and/or diagnose inflamatorydisorders.

In another specific embodiment, anti-D-SLAM antibodies of the inventionare used to treat, prevent, modulate, detect, and/or diagnose allergyand/or hypersensitivity.

In a specific embodiment, D-SLAM polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, are used to treat,prevent, and/or diagnose chronic obstructive pulmonary disease (COPD).

In another embodiment, D-SLAM polynucleotides or polypeptides of theinvention and/or agonists and/or antagonists thereof, are used to treat,prevent, and/or diagnose fibroses and conditions associated withfibroses, such as, for example, but not limited to, cystic fibrosis(including such fibroses as cystic fibrosis of the pancreas,Clarke-Hadfield syndrome, fibrocystic disease of the pancreas,mucoviscidosis, and viscidosis), endomyocardial fibrosis, idiopathicretroperitoneal fibrosis, leptomeningeal fibrosis, mediastinal fibrosis,nodular subepidermal fibrosis, pericentral fibrosis, perimuscularfibrosis, pipestem fibrosis, replacement fibrosis, subadventitialfibrosis, and Symmers' clay pipestem fibrosis.

Diseases associated with increased cell survival, or the inhibition ofapoptosis that may be diagnosed, treated, or prevented with the D-SLAMpolynucleotides or polypeptides of the invention, and agonists andantagonists thereof, include cancers (such as follicular lymphomas,carcinomas with p53 mutations, and hormone-dependent tumors, including,but not limited to, colon cancer, cardiac tumors, pancreatic cancer,melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as systemiclupus erythematosus and immune-related glomerulonephritis rheumatoidarthritis); viral infections (such as herpes viruses, pox viruses andadenoviruses); inflammation; graft vs. host disease; acute graftrejection and chronic graft rejection. Thus, in preferred embodimentsD-SLAM polynucleotides or polypeptides of the invention abd/or agonistsor antagonists thereof, are used to treat, prevent, and/or diagnoseautoimmune diseases and/or inhibit the growth, progression, and/ormetastasis of cancers, including, but not limited to, those cancersdisclosed herein, such as, for example, lymphocytic leukemias(including, for example, MLL and chronic lymphocytic leukemia (CLL)) andfollicular lymphomas. In another embodiment D-SLAM polynucleotides orpolypeptides of the invention are used to activate, differentiate orproliferate cancerous cells or tissue (e.g., B cell lineage relatedcancers (e.g., CLL and MLL), lymphocytic leukemia, or lymphoma) andthereby render the cells more vulnerable to cancer therapy (e.g.,chemotherapy or radiation therapy).

Moreover, in other embodiments, D-SLAM polynucleotides or polypeptidesof the invention or agonists or antagonists thereof, are used to inhibitthe growth, progression, and/or metastases of malignancies and relateddisorders such as leukemia (including acute leukemias (e.g., acutelymphocytic leukemia, acute myelocytic leukemia (including myeloblastic,promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) andchronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia andchronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.,Hodgkin's disease and non-Hodgkin's disease), multiple myeloma,Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumorsincluding, but not limited to, sarcomas and carcinomas such asfibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma, andretinoblastoma.

Diseases associated with increased apoptosis apoptosis that may bediagnosed, treated, or prevented with the D-SLAM polynucleotides orpolypeptides of the invention, and agonists and antagonists thereof,include AIDS; neurodegenerative disorders (such as Alzheimer's disease,Parkinson's disease, Amyotrophic lateral sclerosis, Retinitispigmentosa, Cerebellar degeneration); myelodysplastic syndromes (such asaplastic anemia), ischemic injury (such as that caused by myocardialinfarction, stroke and reperfusion injury), toxin-induced liver disease(such as that caused by alcohol), septic shock, cachexia and anorexia.Thus, in preferred embodiments D-SLAM polynucleotides or polypeptides ofthe invention and/or agonists or antagonists thereof, are used to treat,prevent, and/or diagnose the diseases and disorders listed above.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in the diagnosis and treatment orprevention of a wide range of diseases and/or conditions. Such diseasesand conditions include, but are not limited to, cancer (e.g., immunecell related cancers, breast cancer, prostate cancer, ovarian cancer,follicular lymphoma, cancer associated with mutation or alteration ofp53, brain tumor, bladder cancer, uterocervical cancer, colon cancer,colorectal cancer, non-small cell carcinoma of the lung, small cellcarcinoma of the lung, stomach cancer, etc.), lymphoproliferativedisorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial,etc.) infection (e.g., HIV-1 infection, HIV-2 infection, herpesvirusinfection (including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6,HHV-7, EBV), adenovirus infection, poxvirus infection, human papillomavirus infection, hepatitis infection (e.g., HAV, HBV, HCV, etc.),Helicobacter pylori infection, invasive Staphylococcia, etc.), parasiticinfection, nephritis, bone disease (e.g., osteoporosis),atherosclerosis, pain, cardiovascular disorders (e.g.,neovascularization, hypovascularization or reduced circulation (e.g.,ischemic disease (e.g., myocardial infarction, stroke, etc.)), A/DS,allergy, inflammation, neurodegenerative disease (e.g., Alzheimer'sdisease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentaryretinitis, cerebellar degeneration, etc.), graft rejection (acute andchronic), graft vs. host disease, diseases due to osteomyelodysplasia(e.g., aplastic anemia, etc.), joint tissue destruction in rheumatism,liver disease (e.g., acute and chronic hepatitis, liver injury, andcirrhosis), autoimmune disease (e.g., multiple sclerosis, rheumatoidarthritis, systemic lupus erythematosus, immune complexglomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenicpurpura, Grave's disease, Hashimoto's thyroiditis, etc.), cardiomyopathy(e.g., dilated cardiomyopathy), diabetes, diabetic complications (e.g.,diabetic nephropathy, diabetic neuropathy, diabetic retinopathy),influenza, asthma, psoriasis, glomerulonephritis, septic shock, andulcerative colitis.

Polynucleotides and/or polypeptides of the invention and/or agonistsand/or antagonists thereof are useful in promoting angiogenesis, woundhealing (e.g., wounds, burns, and bone fractures). Polynucleotidesand/or polypeptides of the invention and/or agonists and/or antagoniststhereof are also useful as an adjuvant to enhance immune responsivenessto specific antigen, anti-viral immune responses.

More generally, polynucleotides and/or polypeptides of the inventionand/or agonists and/or antagonists thereof are useful in regulating(i.e., elevating or reducing) immune response. For example,polynucleotides and/or polypeptides of the invention may be useful inpreparation or recovery from surgery, trauma, radiation therapy,chemotherapy, and transplantation, or may be used to boost immuneresponse and/or recovery in the elderly and immunocompromisedindividuals. Alternatively, polynucleotides and/or polypeptides of theinvention and/or agonists and/or antagonists thereof are useful asimmunosuppressive agents, for example in the treatment or prevention ofautoimmune disorders. In specific embodiments, polynucleotides and/orpolypeptides of the invention are used to treat or prevent chronicinflammatory, allergic or autoimmune conditions, such as those describedherein or are otherwise known in the art.

Preferably, treatment using D-SLAM polynucleotides or polypeptides,and/or agonists or antagonists of D-SLAM (e.g., anti-D-SLAM antibody),could either be by administering an effective amount of D-SLAMpolypeptide of the invention, or agonist or antagonist thereof, to thepatient, or by removing cells from the patient, supplying the cells withD-SLAM polynucleotide, and returning the engineered cells to the patient(ex vivo therapy). Moreover, as further discussed herein, the D-SLAMpolypeptide or polynucleotide can be used as an adjuvant in a vaccine toraise an immune response against infectious disease.

Immune Activity

In one embodiment, D-SLAM polynucleotides or polypeptides or D-SLAMagonists or antagonists (e.g., anti-D-SLAM antibodies) of the inventionare used to treat, prevent, diagnose, or prognose an individual havingan immunodeficiency.

Immunodeficiencies that may be treated, prevented, diagnosed, and/orprognosed with the D-SLAM polynucleotides or polypeptides or D-SLAMagonists or antagonists (e.g., anti-D-SLAM antibodies) of the invention,include, but are not limited to one or more immunodeficiencies selectedfrom: severe combined immunodeficiency (SCID)-X linked, SCID-autosomal,adenosine deaminase deficiency (ADA deficiency), X-linkedagammaglobulinemia (XLA), Bruton's disease, congenitalagammaglobulinemia, X-linked infantile agammaglobulinemia, acquiredagammaglobulinemia, adult onset agammaglobulinemia, late-onsetagammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia,transient hypogammaglobulinemia of infancy, unspecifiedhypogammaglobulinemia, agammaglobulinemia, common variableimmunodeficiency (CVID) (acquired), Wiskott-Aldrich Syndrome (WAS),X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiencywith hyper IgM, selective IgA deficiency, IgG subclass deficiency (withor without IgA deficiency), antibody deficiency with normal or elevatedIgs, immunodeficiency with thymoma, Ig heavy chain deletions, kappachain deficiency, B cell lymphoproliferative disorder (BLPD), selectiveIgM immunodeficiency, recessive agammaglobulinemia (Swiss type),reticular dysgenesis, neonatal neutropenia, severe congenitalleukopenia, thymic alymphoplasia-aplasia or dysplasia withimmunodeficiency, ataxia-telangiectasia, short limbed dwarfism, X-linkedlymphoproliferative syndrome (XLP), Nezelof syndrome-combinedimmunodeficiency with Igs, purine nucleoside phosphorylase deficiency(PNP), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severecombined immunodeficiency.

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, may be useful in treating, diagnosing, detecting, and/orpreventing deficiencies or disorders of the immune system, by activatingor inhibiting the proliferation, differentiation, or mobilization(chemotaxis) of immune cells. Immune cells develop through a processcalled hematopoiesis, producing myeloid (platelets, red blood cells,neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cellsfrom pluripotent stem cells. The etiology of these immune deficienciesor disorders may be genetic, somatic, such as cancer or some autoimmunedisorders, acquired (e.g., by chemotherapy or toxins), or infectious.Moreover, D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can be used as a marker or detector of aparticular immune system disease or disorder.

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, may be useful in treating, diagnosing, detecting, and/orpreventing deficiencies or disorders of hematopoietic cells. D-SLAMpolynucleotides or polypeptides, or agonists or antagonists of D-SLAM,could be used to increase differentiation and proliferation ofhematopoietic cells, including the pluripotent stem cells, in an effortto treat, diagnose, detect, and/or prevent those disorders associatedwith a decrease in certain (or many) types hematopoietic cells. Examplesof immunologic deficiency syndromes include, but are not limited to:blood protein disorders (e.g. agammaglobulinemia, dysgammaglobulinemia),ataxia telangiectasia, common variable immunodeficiency, DigeorgeSyndrome, HIV infection, HTLV-BLV infection, leukocyte adhesiondeficiency syndrome, lymphopenia, phagocyte bactericidal dysfunction,severe combined immunodeficiency (SCIDs), Wiskott-Aldrich Disorder,anemia, thrombocytopenia, or hemoglobinuria.

Moreover, D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can also be used to modulate hemostatic (thestopping of bleeding) or thrombolytic activity (clot formation). Forexample, by increasing hemostatic or thrombolytic activity, D-SLAMpolynucleotides or polypeptides, or agonists or antagonists of D-SLAM,could be used to treat, diagnose, detect, and/or prevent bloodcoagulation disorders (e.g., afibrinogenemia, factor deficiencies),blood platelet disorders (e.g. thrombocytopenia), or wounds resultingfrom trauma, surgery, or other causes. Alternatively, D-SLAMpolynucleotides or polypeptides, or agonists or antagonists of D-SLAM,that can decrease hemostatic or thrombolytic activity could be used toinhibit or dissolve clotting, important in the treatment, diagnosis,detection, and/or prevention of heart attacks (infarction), strokes, orscarring.

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, may also be useful in treating, diagnosing, detecting, and/orpreventing autoimmune disorders. Many autoimmune disorders result frominappropriate recognition of self as foreign material by immune cells.This inappropriate recognition results in an immune response leading tothe destruction of the host tissue. Therefore, the administration ofD-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, that can inhibit an immune response, particularly theproliferation, differentiation, or chemotaxis of T-cells, may be aneffective therapy in preventing autoimmune disorders.

Examples of autoimmune disorders that can be treated, diagnosed,detected, and/or prevented include, but are not limited to: Addison'sDisease, hemolytic anemia, antiphospholipid syndrome, rheumatoidarthritis, dermatitis, allergic encephalomyelitis, glomerulonephritis,Goodpasture's Syndrome, Graves' Disease, Multiple Sclerosis, MyastheniaGravis, Neuritis, Ophthalmia, Bullous Pemphigoid, Pemphigus,Polyendocrinopathies, Purpura, Reiter's Disease, Stiff-Man Syndrome,Autoimmune Thyroiditis, Systemic Lupus Erythematosus, AutoimmunePulmonary Inflammation, Guillain-Barre Syndrome, insulin dependentdiabetes mellitis, and autoimmune inflammatory eye disease.

Similarly, allergic reactions and conditions, such as asthma(particularly allergic asthma) or other respiratory problems, may alsobe treated, diagnosed, detected, and/or prevented by D-SLAMpolynucleotides or polypeptides, or agonists or antagonists of D-SLAM.Moreover, these molecules can be used to treat, diagnose, detect, and/orprevent anaphylaxis, hypersensitivity to an antigenic molecule, or bloodgroup incompatibility.

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, may also be used to treat, diagnose, detect, and/or preventorgan rejection or graft-versus-host disease (GVHD). Organ rejectionoccurs by host immune cell destruction of the transplanted tissuethrough an immune response. Similarly, an immune response is alsoinvolved in GVHD, but, in this case, the foreign transplanted immunecells destroy the host tissues. The administration of D-SLAMpolynucleotides or polypeptides, or agonists or antagonists of D-SLAM,that inhibits an immune response, particularly the proliferation,differentiation, or chemotaxis of T-cells, may be an effective therapyin preventing organ rejection or GVHD.

Similarly, D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may also be used to modulate inflammation. Forexample, D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may inhibit the proliferation and differentiationof cells involved in an inflammatory response. These molecules can beused to treat, diagnose, detect, and/or prevent inflammatory conditions,both chronic and acute conditions, including inflammation associatedwith infection (e.g., septic shock, sepsis, or systemic inflammatoryresponse syndrome (SIRS)), ischemia-reperfusion injury, endotoxinlethality, arthritis, complement-mediated hyperacute rejection,nephritis, cytokine or chemokine induced lung injury, inflammatory boweldisease, Crohn's disease, or resulting from over production of cytokines(e.g., TNF or IL-1.)

Hyperproliferative Disorders

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, can be used to treat, diagnose, detect, and/or preventhyperproliferative disorders, including neoplasms. D-SLAMpolynucleotides or polypeptides, or agonists or antagonists of D-SLAM,may inhibit the proliferation of the disorder through direct or indirectinteractions. Alternatively, D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, may proliferate other cells which caninhibit the hyperproliferative disorder.

For example, by increasing an immune response, particularly increasingantigenic qualities of the hyperproliferative disorder or byproliferating, differentiating, or mobilizing T-cells,hyperproliferative disorders can be treated, diagnosed, detected, and/orprevented. This immune response may be increased by either enhancing anexisting immune response, or by initiating a new immune response.Alternatively, decreasing an immune response may also be a method oftreating, diagnosing, detecting, and/or preventing hyperproliferativedisorders, such as a chemotherapeutic agent.

Examples of hyperproliferative disorders that can be treated, diagnosed,detected, and/or prevented by D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, include, but are not limited toneoplasms located in the: abdomen, bone, breast, digestive system,liver, pancreas, peritoneum, endocrine glands (adrenal, parathyroid,pituitary, testicles, ovary, thymus, thyroid), eye, head and neck,nervous (central and peripheral), lymphatic system, pelvic, skin, softtissue, spleen, thoracic, and urogenital.

Similarly, other hyperproliferative disorders can also be treated,diagnosed, detected, and/or prevented by D-SLAM polynucleotides orpolypeptides, or agonists or antagonists of D-SLAM. Examples of suchhyperproliferative disorders include, but are not limited to:hypergammaglobulinemia, lymphoproliferative disorders, paraproteinemias,purpura, sarcoidosis, Sezary Syndrome, Waldenstron's Macroglobulinemia,Gaucher's Disease, histiocytosis, and any other hyperproliferativedisease, besides neoplasia, located in an organ system listed above.

Cardiovascular Disorders

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, encoding D-SLAM may be used to treat, diagnose, detect, and/orprevent cardiovascular disorders, including peripheral artery disease,such as limb ischemia.

Cardiovascular disorders include cardiovascular abnormalities, such asarterio-arterial fistula, arteriovenous fistula, cerebral arteriovenousmalformations, congenital heart defects, pulmonary atresia, and ScimitarSyndrome. Congenital heart defects include aortic coarctation, cortriatriatum, coronary vessel anomalies, crisscross heart, dextrocardia,patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex,hypoplastic left heart syndrome, levocardia, tetralogy of fallot,transposition of great vessels, double outlet right ventricle, tricuspidatresia, persistent truncus arteriosus, and heart septal defects, suchas aortopulmonary septal defect, endocardial cushion defects,Lutembacher's Syndrome, trilogy of Fallot, ventricular heart septaldefects.

Cardiovascular disorders also include heart disease, such asarrhythmias, carcinoid heart disease, high cardiac output, low cardiacoutput, cardiac tamponade, endocarditis (including bacterial), heartaneurysm, cardiac arrest, congestive heart failure, congestivecardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy,congestive cardiomyopathy, left ventricular hypertrophy, rightventricular hypertrophy, post-infarction heart rupture, ventricularseptal rupture, heart valve diseases, myocardial diseases, myocardialischemia, pericardial effusion, pericarditis (including constrictive andtuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonaryheart disease, rheumatic heart disease, ventricular dysfunction,hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome,cardiovascular syphilis, and cardiovascular tuberculosis.

Arrhythmias include sinus arrhythmia, atrial fibrillation, atrialflutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branchblock, sinoatrial block, long QT syndrome, parasystole,Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome,Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, andventricular fibrillation. Tachycardias include paroxysmal tachycardia,supraventricular tachycardia, accelerated idioventricular rhythm,atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia,ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia,sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.

Heart valve disease include aortic valve insufficiency, aortic valvestenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse,tricuspid valve prolapse, mitral valve insufficiency, mitral valvestenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonaryvalve stenosis, tricuspid atresia, tricuspid valve insufficiency, andtricuspid valve stenosis.

Myocardial diseases include alcoholic cardiomyopathy, congestivecardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvularstenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy,Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardialfibrosis, Kearns Syndrome, myocardial reperfusion injury, andmyocarditis.

Myocardial ischemias include coronary disease, such as angina pectoris,coronary aneurysm, coronary arteriosclerosis, coronary thrombosis,coronary vasospasm, myocardial infarction and myocardial stunning.

Cardiovascular diseases also include vascular diseases such asaneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis,Hippel-Lindau Disease, Klippel-Trenaunay-Weber Syndrome, Sturge-WeberSyndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis,aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis,enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabeticangiopathies, diabetic retinopathy, embolisms, thrombosis,erythromelalgia, hemorrhoids, hepatic veno-occlusive disease,hypertension, hypotension, ischemia, peripheral vascular diseases,phlebitis, pulmonary veno-occlusive disease, Raynaud's disease, CRESTsyndrome, retinal vein occlusion, Scimitar syndrome, superior vena cavasyndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagictelangiectasia, varicocele, varicose veins, varicose ulcer, vasculitis,and venous insufficiency.

Aneurysms include dissecting aneurysms, false aneurysms, infectedaneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms,coronary aneurysms, heart aneurysms, and iliac aneurysms.

Arterial occlusive diseases include arteriosclerosis, intermittentclaudication, carotid stenosis, fibromuscular dysplasias, mesentericvascular occlusion, Moyamoya disease, renal artery obstruction, retinalartery occlusion, and thromboangiitis obliterans.

Cerebrovascular disorders include carotid artery diseases, cerebralamyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebralarteriosclerosis, cerebral arteriovenous malformation, cerebral arterydiseases, cerebral embolism and thrombosis, carotid artery thrombosis,sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epiduralhematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebralinfarction, cerebral ischemia (including transient), subclavian stealsyndrome, periventricular leukomalacia, vascular headache, clusterheadache, migraine, and vertebrobasilar insufficiency.

Embolisms include air embolisms, amniotic fluid embolisms, cholesterolembolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, andthromoboembolisms. Thrombosis include coronary thrombosis, hepatic veinthrombosis, retinal vein occlusion, carotid artery thrombosis, sinusthrombosis, Wallenberg's syndrome, and thrombophlebitis.

Ischemia includes cerebral ischemia, ischemic colitis, compartmentsyndromes, anterior compartment syndrome, myocardial ischemia,reperfusion injuries, and peripheral limb ischemia. Vasculitis includesaortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome,mucocutaneous lymph node syndrome, thromboangiitis obliterans,hypersensitivity vasculitis, Schoenlein-Henoch purpura, allergiccutaneous vasculitis, and Wegener's granulomatosis.

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, are especially effective for the treatment, diagnosis,detection, and/or prevention of critical limb ischemia and coronarydisease. As shown in the Examples, administration of D-SLAMpolynucleotides and polypeptides to an experimentally induced ischemiarabbit hindlimb may restore blood pressure ratio, blood flow,angiographic score, and capillary density.

D-SLAM polypeptides may be administered using any method known in theart, including, but not limited to, direct needle injection at thedelivery site, intravenous injection, topical administration, catheterinfusion, biolistic injectors, particle accelerators, gelfoam spongedepots, other commercially available depot materials, osmotic pumps,oral or suppositorial solid pharmaceutical formulations, decanting ortopical applications during surgery, aerosol delivery. Such methods areknown in the art. D-SLAM polypeptides may be administered as part of apharmaceutical composition, described in more detail below. Methods ofdelivering D-SLAM polynucleotides are described in more detail herein.

Anti-Angiogenesis Activity

The naturally occurring balance between endogenous stimulators andinhibitors of angiogenesis is one in which inhibitory influencespredominate. Rastinejad et al., Cell 56:345-355 (1989). In those rareinstances in which neovascularization occurs under normal physiologicalconditions, such as wound healing, organ regeneration, embryonicdevelopment, and female reproductive processes, angiogenesis isstringently regulated and spatially and temporally delimited. Underconditions of pathological angiogenesis such as that characterizingsolid tumor growth, these regulatory controls fail. Unregulatedangiogenesis becomes pathologic and sustains progression of manyneoplastic and non-neoplastic diseases. A number of serious diseases aredominated by abnormal neovascularization including solid tumor growthand metastases, arthritis, some types of eye disorders, and psoriasis.See, e.g., reviews by Moses et al., Biotech. 9:630-634 (1991); Folkmanet al., N. Engl. J. Med., 333:1757-1763 (1995); Auerbach et al., J.Microvasc. Res. 29:401-411 (1985); Folkman, Advances in Cancer Research,eds. Klein and Weinhouse, Academic Press, New York, pp. 175-203 (1985);Patz, Am. J. Opthalmol. 94:715-743 (1982); and Folkman et al., Science221:719-725 (1983). In a number of pathological conditions, the processof angiogenesis contributes to the disease state. For example,significant data have accumulated which suggest that the growth of solidtumors is dependent on angiogenesis. Folkman and Klagsbrun, Science235:442-447 (1987).

The present invention provides for treatment, diagnosis, detection,and/or prevention of diseases or disorders associated withneovascularization by administration of the D-SLAM polynucleotidesand/or polypeptides of the invention, as well as agonists or antagonistsof D-SLAM. Malignant and metastatic conditions which can be treated,diagnosed, detected, and/or prevented with the polynucleotides andpolypeptides, or agonists or antagonists of the invention include, butare not limited to, malignancies, solid tumors, and cancers describedherein and otherwise known in the art (for a review of such disorders,see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia(1985)):

Ocular disorders associated with neovascularization which can betreated, diagnosed, detected, and/or prevented with the D-SLAMpolynucleotides and polypeptides of the present invention (includingD-SLAM agonists and/or antagonists) include, but are not limited to:neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolentalfibroplasia, uveitis, retinopathy of prematurity macular degeneration,corneal graft neovascularization, as well as other eye inflammatorydiseases, ocular tumors and diseases associated with choroidal or irisneovascularization. See, e.g., reviews by Waltman et al., Am. J.Ophthal. 85:704-710 (1978) and Gartner et al., Surv. Ophthal. 22:291-312(1978).

Additionally, disorders which can be treated, diagnosed, detected,and/or prevented with the D-SLAM polynucleotides and polypeptides of thepresent invention (including D-SLAM agonist and/or antagonists) include,but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma,atherosclerotic plaques, delayed wound healing, granulations, hemophilicjoints, hypertrophic scars, nonunion fractures, Osler-Weber syndrome,pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.

Moreover, disorders and/or states, which can be treated, diagnosed,detected, and/or prevented with the D-SLAM polynucleotides andpolypeptides of the present invention (including D-SLAM agonist and/orantagonists) include, but are not limited to, solid tumors, blood borntumors such as leukemias, tumor metastasis, Kaposi's sarcoma, benigntumors, for example hemangiomas, acoustic neuromas, neurofibromas,trachomas, and pyogenic granulomas, rheumatoid arthritis, psoriasis,ocular angiogenic diseases, for example, diabetic retinopathy,retinopathy of prematurity, macular degeneration, corneal graftrejection, neovascular glaucoma, retrolental fibroplasia, rubeosis,retinoblastoma, and uvietis, delayed wound healing, endometriosis,vascluogenesis, granulations, hypertrophic scars (keloids), nonunionfractures, scleroderma, trachoma, vascular adhesions, myocardialangiogenesis, coronary collaterals, cerebral collaterals, arteriovenousmalformations, ischemic limb angiogenesis, Osler-Webber Syndrome, plaqueneovascularization, telangiectasia, hemophiliac joints, angiofibromafibromuscular dysplasia, wound granulation, Crohn's disease,atherosclerosis, birth control agent by preventing vascularizationrequired for embryo implantation controlling menstruation, diseases thathave angiogenesis as a pathologic consequence such as cat scratchdisease (Rochele minalia quintosa), ulcers (Helicobacter pylori),Bartonellosis and bacillary angiomatosis.

Diseases at the Cellular Level

Diseases associated with increased cell survival or the inhibition ofapoptosis that could be treated, diagnosed, detected, and/or preventedby D-SLAM polynucleotides or polypeptides, as well as antagonists oragonists of D-SLAM, include cancers (such as follicular lymphomas,carcinomas with p53 mutations, and hormone-dependent tumors, including,but not limited to colon cancer, cardiac tumors, pancreatic cancer,melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer,testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma,lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi'ssarcoma and ovarian cancer); autoimmune disorders (such as, multiplesclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliarycirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemiclupus erythematosus and immune-related glomerulonephritis and rheumatoidarthritis) and viral infections (such as herpes viruses, pox viruses andadenoviruses), inflammation, graft v. host disease, acute graftrejection, and chronic graft rejection. In preferred embodiments, D-SLAMpolynucleotides, polypeptides, and/or antagonists of the invention areused to inhibit growth, progression, and/or metasis of cancers, inparticular those listed above.

Additional diseases or conditions associated with increased cellsurvival that could be treated, diagnosed, detected, and/or prevented byD-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, include, but are not limited to, progression, and/or metastasesof malignancies and related disorders such as leukemia (including acuteleukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia(including myeloblastic, promyelocytic, myelomonocytic, monocytic, anderythroleukemia)) and chronic leukemias (e.g., chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemiavera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease),multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease,and solid tumors including, but not limited to, sarcomas and carcinomassuch as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma,osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma,lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma,Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma,pancreatic cancer, breast cancer, ovarian cancer, prostate cancer,squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweatgland carcinoma, sebaceous gland carcinoma, papillary carcinoma,papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma,bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile ductcarcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor,cervical cancer, testicular tumor, lung carcinoma, small cell lungcarcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma,melanoma, neuroblastoma, and retinoblastoma.

Diseases associated with increased apoptosis that could be treated,diagnosed, detected, and/or prevented by D-SLAM polynucleotides orpolypeptides, as well as agonists or antagonists of D-SLAM, includeAIDS; neurodegenerative disorders (such as Alzheimer's disease,Parkinson's disease, Amyotrophic lateral sclerosis, Retinitispigmentosa, Cerebellar degeneration and brain tumor or prior associateddisease); autoimmune disorders (such as, multiple sclerosis, Sjogren'ssyndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease,Crohn's disease, polymyositis, systemic lupus erythematosus andimmune-related glomerulonephritis and rheumatoid arthritis)myelodysplastic syndromes (such as aplastic anemia), graft v. hostdisease, ischemic injury (such as that caused by myocardial infarction,stroke and reperfusion injury), liver injury (e.g., hepatitis relatedliver injury, ischemia/reperfusion injury, cholestosis (bile ductinjury) and liver cancer); toxin-induced liver disease (such as thatcaused by alcohol), septic shock, cachexia and anorexia.

Wound Healing and Epithelial Cell Proliferation

In accordance with yet a further aspect of the present invention, thereis provided a process for utilizing D-SLAM polynucleotides orpolypeptides, as well as agonists or antagonists of D-SLAM, fortherapeutic purposes, for example, to stimulate epithelial cellproliferation and basal keratinocytes for the purpose of wound healing,and to stimulate hair follicle production and healing of dermal wounds.D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, may be clinically useful in stimulating woundhealing including surgical wounds, excisional wounds, deep woundsinvolving damage of the dermis and epidermis, eye tissue wounds, dentaltissue wounds, oral cavity wounds, diabetic ulcers, dermal ulcers,cubitus ulcers, arterial ulcers, venous stasis ulcers, burns resultingfrom heat exposure or chemicals, and other abnormal wound healingconditions such as uremia, malnutrition, vitamin deficiencies andcomplications associted with systemic treatment with steroids, radiationtherapy and antineoplastic drugs and antimetabolites. D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, could be used to promote dermal reestablishment subsequent todermal loss

D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could be used to increase the adherence of skingrafts to a wound bed and to stimulate re-epithelialization from thewound bed. The following are types of grafts that D-SLAM polynucleotidesor polypeptides, agonists or antagonists of D-SLAM, could be used toincrease adherence to a wound bed: autografts, artificial skin,allografts, autodermic graft, autoepdermic grafts, avacular grafts,Blair-Brown grafts, bone graft, brephoplastic grafts, cutis graft,delayed graft, dermic graft, epidermic graft, fascia graft, fullthickness graft, heterologous graft, xenograft, homologous graft,hyperplastic graft, lamellar graft, mesh graft, mucosal graft,Ollier-Thiersch graft, omenpal graft, patch graft, pedicle graft,penetrating graft, split skin graft, thick split graft. D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, can be used to promote skin strength and to improve theappearance of aged skin.

It is believed that D-SLAM polynucleotides or polypeptides, as well asagonists or antagonists of D-SLAM, will also produce changes inhepatocyte proliferation, and epithelial cell proliferation in the lung,breast, pancreas, stomach, small intesting, and large intestine. D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, could promote proliferation of epithelial cells such assebocytes, hair follicles, hepatocytes, type II pneumocytes,mucin-producing goblet cells, and other epithelial cells and theirprogenitors contained within the skin, lung, liver, and gastrointestinaltract. D-SLAM polynucleotides or polypeptides, agonists or antagonistsof D-SLAM, may promote proliferation of endothelial cells,keratinocytes, and basal keratinocytes.

D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could also be used to reduce the side effects ofgut toxicity that result from radiation, chemotherapy treatments orviral infections. D-SLAM polynucleotides or polypeptides, as well asagonists or antagonists of D-SLAM, may have a cytoprotective effect onthe small intestine mucosa. D-SLAM polynucleotides or polypeptides, aswell as agonists or antagonists of D-SLAM, may also stimulate healing ofmucositis (mouth ulcers) that result from chemotherapy and viralinfections.

D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could further be used in full regeneration ofskin in full and partial thickness skin defects, including burns, (i.e.,repopulation of hair follicles, sweat glands, and sebaceous glands),treatment, diagnosis, detection, and/or prevention of other skin defectssuch as psoriasis. D-SLAM polynucleotides or polypeptides, as well asagonists or antagonists of D-SLAM, could be used to treat, diagnose,detect, and/or prevent epidermolysis bullosa, a defect in adherence ofthe epidermis to the underlying dermis which results in frequent, openand painful blisters by accelerating reepithelialization of theselesions. D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could also be used to treat, diagnose, detect,and/or prevent gastric and doudenal ulcers and help heal by scarformation of the mucosal lining and regeneration of glandular mucosa andduodenal mucosal lining more rapidly. Inflamamatory bowel diseases, suchas Crohn's disease and ulcerative colitis, are diseases which result indestruction of the mucosal surface of the small or large intestine,respectively. Thus, D-SLAM polynucleotides or polypeptides, as well asagonists or antagonists of D-SLAM, could be used to promote theresurfacing of the mucosal surface to aid more rapid healing and toprevent progression of inflammatory bowel disease. Treatment, diagnosis,detection, and/or prevention with D-SLAM polynucleotides orpolypeptides, agonists or antagonists of D-SLAM, is expected to have asignificant effect on the production of mucus throughout thegastrointestinal tract and could be used to protect the intestinalmucosa from injurious substances that are ingested or following surgery.D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could be used to treat, diagnose, detect, and/orprevent diseases associate with the under expression of D-SLAM.

Moreover, D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could be used to prevent and heal damage to thelungs due to various pathological states. A growth factor such as D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, which could stimulate proliferation and differentiation andpromote the repair of alveoli and brochiolar epithelium to prevent ortreat, diagnose, detect, and/or prevent acute or chronic lung damage.For example, emphysema, which results in the progressive loss of aveoli,and inhalation injuries, i.e., resulting from smoke inhalation andburns, that cause necrosis of the bronchiolar epithelium and alveolicould be effectively treated, diagnosed, detected, and/or preventedusing D-SLAM polynucleotides or polypeptides, agonists or antagonists ofD-SLAM. Also, D-SLAM polynucleotides or polypeptides, as well asagonists or antagonists of D-SLAM, could be used to stimulate theproliferation of and differentiation of type II pneumocytes, which mayhelp treat, diagnose, detect, and/or prevent disease such as hyalinemembrane diseases, such as infant respiratory distress syndrome andbronchopulmonary displasia, in premature infants.

D-SLAM polynucleotides or polypeptides, as well as agonists orantagonists of D-SLAM, could stimulate the proliferation anddifferentiation of hepatocytes and, thus, could be used to alleviate,treat, diagnose, detect, and/or prevent liver diseases and pathologiessuch as fulminant liver failure caused by cirrhosis, liver damage causedby viral hepatitis and toxic substances (i.e., acetaminophen, carbontetraholoride and other hepatotoxins known in the art).

In addition, D-SLAM polynucleotides or polypeptides, as well as agonistsor antagonists of D-SLAM, could be used treat, diagnose, detect, and/orprevent the onset of diabetes mellitus. In patients with newly diagnosedTypes I and II diabetes, where some islet cell function remains, D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, could be used to maintain the islet function so as to alleviate,delay or prevent permanent manifestation of the disease. Also, D-SLAMpolynucleotides or polypeptides, as well as agonists or antagonists ofD-SLAM, could be used as an auxiliary in islet cell transplantation toimprove or promote islet cell function.

Infectious Disease

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, can be used to treat, diagnose, detect, and/or preventinfectious agents. For example, by increasing the immune response,particularly increasing the proliferation and differentiation of Band/or T cells, infectious diseases may be treated, diagnosed, detected,and/or prevented. The immune response may be increased by eitherenhancing an existing immune response, or by initiating a new immuneresponse. Alternatively, D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, may also directly inhibit theinfectious agent, without necessarily eliciting an immune response.

Viruses are one example of an infectious agent that can cause disease orsymptoms that can be treated, diagnosed, detected, and/or prevented byD-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM. Examples of viruses, include, but are not limited to thefollowing DNA and RNA viral families: Arbovirus, Adenoviridae,Arenaviridae, Arterivirus, Birnaviridae, Bunyaviridae, Caliciviridae,Circoviridae, Coronaviridae, Flaviviridae, Hepadnaviridae (Hepatitis),Herpesviridae (such as, Cytomegalovirus, Herpes Simplex, Herpes Zoster),Mononegavirus (e.g., Paramyxoviridae, Morbillivirus, Rhabdoviridae),Orthomyxoviridae (e.g., Influenza), Papovaviridae, Parvoviridae,Picornaviridae, Poxyiridae (such as Smallpox or Vaccinia), Reoviridae(e.g., Rotavirus), Retroviridae (HTLV-I, HTLV-II, Lentivirus), andTogaviridae (e.g., Rubivirus). Viruses falling within these families cancause a variety of diseases or symptoms, including, but not limited to:arthritis, bronchiollitis, encephalitis, eye infections (e.g.,conjunctivitis, keratitis), chronic fatigue syndrome, hepatitis (A, B,C, E, Chronic Active, Delta), meningitis, opportunistic infections(e.g., AIDS), pneumonia, Burkitt's Lymphoma, chickenpox, hemorrhagicfever, Measles, Mumps, Parainfluenza, Rabies, the common cold, Polio,leukemia, Rubella, sexually transmitted diseases, skin diseases (e.g.,Kaposi's, warts), and viremia. D-SLAM polynucleotides or polypeptides,or agonists or antagonists of D-SLAM, can be used to treat, diagnose,detect, and/or prevent any of these symptoms or diseases.

Similarly, bacterial or fungal agents that can cause disease or symptomsand that can be treated, diagnosed, detected, and/or prevented by D-SLAMpolynucleotides or polypeptides, or agonists or antagonists of D-SLAM,include, but not limited to, the following Gram-Negative andGram-positive bacterial families and fungi: Actinomycetales (e.g.,Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae(e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella,Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis,Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella,Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter,Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriaceae(e.g., Acinetobacter, Gonorrhea, Menigococcal), PasteurellaceaInfections (e.g., Actinobacillus, Heamophilus, Pasteurella),Pseudomonas, Rickettsiaceae, Chlamydiaceae, Syphilis, andStaphylococcal. These bacterial or fungal families can cause thefollowing diseases or symptoms, including, but not limited to:bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis,uveitis), gingivitis, opportunistic infections (e.g., AIDS relatedinfections), paronychia, prosthesis-related infections, Reiter'sDisease, respiratory tract infections, such as Whooping Cough orEmpyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery,Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea,meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis,Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, RheumaticFever, Scarlet Fever, sexually transmitted diseases, skin diseases(e.g., cellulitis, dermatocycoses), toxemia, urinary tract infections,wound infections. D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can be used to treat, diagnose, detect, and/orprevent any of these symptoms or diseases.

Moreover, parasitic agents causing disease or symptoms that can betreated, diagnosed, detected, and/or prevented by D-SLAM polynucleotidesor polypeptides, or agonists or antagonists of D-SLAM, include, but notlimited to, the following families: Amebiasis, Babesiosis, Coccidiosis,Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis,Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis,Trypanosomiasis, and Trichomonas. These parasites can cause a variety ofdiseases or symptoms, including, but not limited to: Scabies,Trombiculiasis, eye infections, intestinal disease (e.g., dysentery,giardiasis), liver disease, lung disease, opportunistic infections(e.g., AIDS related), Malaria, pregnancy complications, andtoxoplasmosis. D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, can be used to treat, diagnose, detect, and/orprevent any of these symptoms or diseases.

Preferably, treatment, diagnosis, detection, and/or prevention usingD-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, could either be by administering an effective amount of D-SLAMpolypeptide to the patient, or by removing cells from the patient,supplying the cells with D-SLAM polynucleotide, and returning theengineered cells to the patient (ex vivo therapy). Moreover, the D-SLAMpolypeptide or polynucleotide can be used as an antigen in a vaccine toraise an immune response against infectious disease.

Regeneration

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, can be used to differentiate, proliferate, and attract cells,leading to the regeneration of tissues. (See, Science 276:59-87 (1997).)The regeneration of tissues could be used to repair, replace, or protecttissue damaged by congenital defects, trauma (wounds, burns, incisions,or ulcers), age, disease (e.g. osteoporosis, osteocarthritis,periodontal disease, liver failure), surgery, including cosmetic plasticsurgery, fibrosis, reperfusion injury, or systemic cytokine damage.

Tissues that could be regenerated using the present invention includeorgans (e.g., pancreas, liver, intestine, kidney, skin, endothelium),muscle (smooth, skeletal or cardiac), vasculature (including vascularand lymphatics), nervous, hematopoietic, and skeletal (bone, cartilage,tendon, and ligament) tissue. Preferably, regeneration occurs without ordecreased scarring. Regeneration also may include angiogenesis.

Moreover, D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may increase regeneration of tissues difficult toheal. For example, increased tendon/ligament regeneration would quickenrecovery time after damage. D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, of the present invention could alsobe used prophylactically in an effort to avoid damage. Specific diseasesthat could be treated, diagnosed, detected, and/or prevented include oftendinitis, carpal tunnel syndrome, and other tendon or ligamentdefects. A further example of tissue regeneration of non-healing woundsincludes pressure ulcers, ulcers associated with vascular insufficiency,surgical, and traumatic wounds.

Similarly, nerve and brain tissue could also be regenerated by usingD-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, to proliferate and differentiate nerve cells. Diseases thatcould be treated, diagnosed, detected, and/or prevented using thismethod include central and peripheral nervous system diseases,neuropathies, or mechanical and traumatic disorders (e.g., spinal corddisorders, head trauma, cerebrovascular disease, and stoke).Specifically, diseases associated with peripheral nerve injuries,peripheral neuropathy (e.g., resulting from chemotherapy or othermedical therapies), localized neuropathies, and central nervous systemdiseases (e.g., Alzheimer's disease, Parkinson's disease, Huntington'sdisease, amyotrophic lateral sclerosis, and Shy-Drager syndrome), couldall be treated, diagnosed, detected, and/or prevented using the D-SLAMpolynucleotides or polypeptides, or agonists or antagonists of D-SLAM.

Chemotaxis

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, may have chemotaxis activity. A chemotaxic molecule attracts ormobilizes cells (e.g., monocytes, fibroblasts, neutrophils, T-cells,mast cells, eosinophils, epithelial and/or endothelial cells) to aparticular site in the body, such as inflammation, infection, or site ofhyperproliferation. The mobilized cells can then fight off and/or healthe particular trauma or abnormality.

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, may increase chemotaxic activity of particular cells. Thesechemotactic molecules can then be used to treat, diagnose, detect,and/or prevent inflammation, infection, hyperproliferative disorders, orany immune system disorder by increasing the number of cells targeted toa particular location in the body. For example, chemotaxic molecules canbe used to treat, diagnose, detect, and/or prevent wounds and othertrauma to tissues by attracting immune cells to the injured location. Asa chemotactic molecule, D-SLAM could also attract fibroblasts, which canbe used to treat, diagnose, detect, and/or prevent wounds.

It is also contemplated that D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, may inhibit chemotactic activity.These molecules could also be used to treat, diagnose, detect, and/orprevent disorders. Thus, D-SLAM polynucleotides or polypeptides, oragonists or antagonists of D-SLAM, could be used as an inhibitor ofchemotaxis.

Binding Activity

D-SLAM polypeptides may be used to screen for molecules that bind toD-SLAM or for molecules to which D-SLAM binds. The binding of D-SLAM andthe molecule may activate (agonist), increase, inhibit (antagonist), ordecrease activity of the D-SLAM or the molecule bound. Examples of suchmolecules include antibodies, oligonucleotides, proteins (e.g.,receptors), or small molecules.

Preferably, the molecule is closely related to the natural ligand ofD-SLAM, e.g., a fragment of the ligand, or a natural substrate, aligand, a structural or functional mimetic. (See, Coligan et al.,Current Protocols in Immunology 1(2):Chapter 5 (1991).) Similarly, themolecule can be closely related to the natural receptor to which D-SLAMbinds, or at least, a fragment of the receptor capable of being bound byD-SLAM (e.g., active site). In either case, the molecule can berationally designed using known techniques.

Preferably, the screening for these molecules involves producingappropriate cells which express D-SLAM, either as a secreted protein oron the cell membrane. Preferred cells include cells from mammals, yeast,Drosophila, or E. coli. Cells expressing D-SLAM (or cell membranecontaining the expressed polypeptide) are then preferably contacted witha test compound potentially containing the molecule to observe binding,stimulation, or inhibition of activity of either D-SLAM or the molecule.

The assay may simply test binding of a candidate compound to D-SLAM,wherein binding is detected by a label, or in an assay involvingcompetition with a labeled competitor. Further, the assay may testwhether the candidate compound results in a signal generated by bindingto D-SLAM.

Alternatively, the assay can be carried out using cell-freepreparations, polypeptide/molecule affixed to a solid support, chemicallibraries, or natural product mixtures. The assay may also simplycomprise the steps of mixing a candidate compound with a solutioncontaining D-SLAM, measuring D-SLAM/molecule activity or binding, andcomparing the D-SLAM/molecule activity or binding to a standard.

Preferably, an ELISA assay can measure D-SLAM level or activity in asample (e.g., biological sample) using a monoclonal or polyclonalantibody. The antibody can measure D-SLAM level or activity by eitherbinding, directly or indirectly, to D-SLAM or by competing with D-SLAMfor a substrate.

Additionally, the receptor to which D-SLAM binds can be identified bynumerous methods known to those of skill in the art, for example, ligandpanning and FACS sorting (Coligan, et al., Current Protocols in Immun.,1(2), Chapter 5, (1991)). For example, expression cloning is employedwherein polyadenylated RNA is prepared from a cell responsive to thepolypeptides, for example, NIH3T3 cells which are known to containmultiple receptors for the FGF family proteins, and SC-3 cells, and acDNA library created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to thepolypeptides. Transfected cells which are grown on glass slides areexposed to the polypeptide of the present invention, after they havebeen labelled. The polypeptides can be labeled by a variety of meansincluding iodination or inclusion of a recognition site for asite-specific protein kinase.

Following fixation and incubation, the slides are subjected toauto-radiographic analysis. Positive pools are identified and sub-poolsare prepared and re-transfected using an iterative sub-pooling andre-screening process, eventually yielding a single clones that encodesthe putative receptor.

As an alternative approach for receptor identification, the labeledpolypeptides can be photoaffinity linked with cell membrane or extractpreparations that express the receptor molecule. Cross-linked materialis resolved by PAGE analysis and exposed to X-ray film. The labeledcomplex containing the receptors of the polypeptides can be excised,resolved into peptide fragments, and subjected to proteinmicrosequencing. The amino acid sequence obtained from microsequencingwould be used to design a set of degenerate oligonucleotide probes toscreen a cDNA library to identify the genes encoding the putativereceptors.

Moreover, the techniques of gene-shuffling, motif-shuffling,exon-shuffling, and/or codon-shuffling (collectively referred to as “DNAshuffling”) may be employed to modulate the activities of D-SLAM therebyeffectively generating agonists and antagonists of D-SLAM. Seegenerally, U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252,and 5,837,458, and Patten, P. A., et al., Curr. Opinion Biotechnol.8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998);Hansson, L. O., et al., J. Mol. Biol. 287:265-76 (1999); and Lorenzo, M.M. and Blasco, R. Biotechniques 24(2):308-13 (1998) (each of thesepatents and publications are hereby incorporated by reference). In oneembodiment, alteration of D-SLAM polynucleotides and correspondingpolypeptides may be achieved by DNA shuffling. DNA shuffling involvesthe assembly of two or more DNA segments into a desired D-SLAM moleculeby homologous, or site-specific, recombination. In another embodiment,D-SLAM polynucleotides and corresponding polypeptides may be alterred bybeing subjected to random mutagenesis by error-prone PCR, randomnucleotide insertion or other methods prior to recombination. In anotherembodiment, one or more components, motifs, sections, parts, domains,fragments, etc., of D-SLAM may be recombined with one or morecomponents, motifs, sections, parts, domains, fragments, etc. of one ormore heterologous molecules. In preferred embodiments, the heterologousmolecules are Secreted Lymphocyte Activation Molecule (SLAM) familymembers. In further preferred embodiments, the heterologous molecule isa growth factor such as, for example, platelet-derived growth factor(PDGF), insulin-like growth factor (IGF-I), transforming growth factor(TGF)-alpha, epidermal growth factor (EGF), fibroblast growth factor(FGF), TGF-beta, bone morphogenetic protein (BMP)-2, BMP-4, BMP-5,BMP-6, BMP-7, activins A and B, decapentaplegic(dpp), 60A, OP-2,dorsalin, growth differentiation factors (GDFs), nodal, MIS,inhibin-alpha, TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta5, andglial-derived neurotrophic factor (GDNF).

Other preferred fragments are biologically active D-SLAM fragments.Biologically active fragments are those exhibiting activity similar, butnot necessarily identical, to an activity of the D-SLAM polypeptide. Thebiological activity of the fragments may include an improved desiredactivity, or a decreased undesirable activity.

Additionally, this invention provides a method of screening compounds toidentify those which modulate the action of the polypeptide of thepresent invention. An example of such an assay comprises combining amammalian fibroblast cell, a the polypeptide of the present invention,the compound to be screened and [³H] thymidine under cell cultureconditions where the fibroblast cell would normally proliferate. Acontrol assay may be performed in the absence of the compound to bescreened and compared to the amount of fibroblast proliferation in thepresence of the compound to determine if the compound stimulatesproliferation by determining the uptake of [³H] thymidine in each case.The amount of fibroblast cell proliferation is measured by liquidscintillation chromatography which measures the incorporation of [³H]thymidine. Both agonist and antagonist compounds may be identified bythis procedure.

In another method, a mammalian cell or membrane preparation expressing areceptor for a polypeptide of the present invention is incubated with alabeled polypeptide of the present invention in the presence of thecompound. The ability of the compound to enhance or block thisinteraction could then be measured. Alternatively, the response of aknown second messenger system following interaction of a compound to bescreened and the D-SLAM receptor is measured and the ability of thecompound to bind to the receptor and elicit a second messenger responseis measured to determine if the compound is a potential agonist orantagonist. Such second messenger systems include but are not limitedto, cAMP guanylate cyclase, ion channels or phosphoinositide hydrolysis.

All of these above assays can be used as diagnostic or prognosticmarkers. The molecules discovered using these assays can be used totreat, diagnose, detect, and/or prevent disease or to bring about aparticular result in a patient (e.g., blood vessel growth) by activatingor inhibiting the D-SLAM/molecule. Moreover, the assays can discoveragents which may inhibit or enhance the production of D-SLAM fromsuitably manipulated cells or tissues. Therefore, the invention includesa method of identifying compounds which bind to D-SLAM comprising thesteps of: (a) incubating a candidate binding compound with D-SLAM; and(b) determining if binding has occurred. Moreover, the inventionincludes a method of identifying agonists/antagonists comprising thesteps of: (a) incubating a candidate compound with D-SLAM, (b) assayinga biological activity, and (b) determining if a biological activity ofD-SLAM has been altered.

Also, one could identify molecules bind D-SLAM experimentally by usingthe beta-pleated sheet regions disclosed in FIG. 3 and Table 1.Accordingly, specific embodiments of the invention are directed topolynucleotides encoding polypeptides which comprise, or alternativelyconsist of, the amino acid sequence of each beta pleated sheet regionsdisclosed in FIG. 3/Table 1. Additional embodiments of the invention aredirected to polynucleotides encoding D-SLAM polypeptides which comprise,or alternatively consist of, any combination or all of the beta pleatedsheet regions disclosed in FIG. 3/Table 1. Additional preferredembodiments of the invention are directed to polypeptides whichcomprise, or alternatively consist of, the D-SLAM amino acid sequence ofeach of the beta pleated sheet regions disclosed in FIG. 3/Table 1.Additional embodiments of the invention are directed to D-SLAMpolypeptides which comprise, or alternatively consist of, anycombination or all of the beta pleated sheet regions disclosed in FIG.3/Table 1.

Antisense and Ribozyme (Antagonists)

In specific embodiments, antagonists according to the present inventionare nucleic acids corresponding to the sequences contained in SEQ IDNO:1, or the complementary strand thereof, and/or to nucleotidesequences contained in the deposited clone 209623. In one embodiment,antisense sequence is generated internally by the organism, in anotherembodiment, the antisense sequence is separately administered (see, forexample, O'Connor, J., Neurochem. 56:560 (1991). Oligodeoxynucleotidesas Anitsense Inhibitors of Gene Expression, CRC Press, Boca Raton, Fla.(1988). Antisense technology can be used to control gene expressionthrough antisense DNA or RNA, or through triple-helix formation.Antisense techniques are discussed for example, in Okano, J., Neurochem.56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of GeneExpression, CRC Press, Boca Raton, Fla. (1988). Triple helix formationis discussed in, for instance, Lee et al., Nucleic Acids Research 6:3073(1979); Cooney et al., Science 241:456 (1988); and Dervan et al.,Science 251:1300 (1991). The methods are based on binding of apolynucleotide to a complementary DNA or RNA.

For example, the 5′ coding portion of a polynucleotide that encodes themature polypeptide of the present invention may be used to design anantisense RNA oligonucleotide of from about 10 to 40 base pairs inlength. A DNA oligonucleotide is designed to be complementary to aregion of the gene involved in transcription thereby preventingtranscription and the production of the receptor. The antisense RNAoligonucleotide hybridizes to the mRNA in vivo and blocks translation ofthe mRNA molecule into receptor polypeptide.

In one embodiment, the D-SLAM antisense nucleic acid of the invention isproduced intracellularly by transcription from an exogenous sequence.For example, a vector or a portion thereof, is transcribed, producing anantisense nucleic acid (RNA) of the invention. Such a vector wouldcontain a sequence encoding the D-SLAM antisense nucleic acid. Such avector can remain episomal or become chromosomally integrated, as longas it can be transcribed to produce the desired antisense RNA. Suchvectors can be constructed by recombinant DNA technology methodsstandard in the art. Vectors can be plasmid, viral, or others know inthe art, used for replication and expression in vertebrate cells.Expression of the sequence encoding D-SLAM, or fragments thereof, can beby any promoter known in the art to act in vertebrate, preferably humancells. Such promoters can be inducible or constitutive. Such promotersinclude, but are not limited to, the SV40 early promoter region(Bernoist and Chambon, Nature 29:304-310 (1981), the promoter containedin the 3′ long terminal repeat of Rous sarcoma virus (Yamamoto et al.,Cell 22:787-797 (1980), the herpes thymidine promoter (Wagner et al.,Proc. Natl. Acad. Sci. U.S.A. 78:1441-1445 (1981), the regulatorysequences of the metallothionein gene (Brinster, et al., Nature296:39-42 (1982)), etc.

The antisense nucleic acids of the invention comprise, or alternativelyconsist of, a sequence complementary to at least a portion of an RNAtranscript of a D-SLAM gene. However, absolute complementarity, althoughpreferred, is not required. A sequence “complementary to at least aportion of an RNA,” referred to herein, means a sequence havingsufficient complementarity to be able to hybridize with the RNA, forminga stable duplex; in the case of double stranded D-SLAM antisense nucleicacids, a single strand of the duplex DNA may thus be tested, or triplexformation may be assayed. The ability to hybridize will depend on boththe of complementarity and the length of the antisense nucleic acidGenerally, the larger the hybridizing nucleic acid, the more basemismatches with a D-SLAM RNA it may contain and still form a stableduplex (or triplex as the case may be). One skilled in the art canascertain a tolerable of mismatch by use of standard procedures todetermine the melting point of the hybridized complex.

Oligonucleotides that are complementary to the 5′ end of the message,e.g., the 5′ untranslated sequence up to and including the AUGinitiation codon, should work most efficiently at inhibitingtranslation. However, sequences complementary to the 3′ untranslatedsequences of mRNAs have been shown to be effective at inhibitingtranslation of mRNAs as well. See generally, Wagner, R., 1994, Nature372:333-335. Thus, oligonucleotides complementary to either the 5′- or3′-non-translated, non-coding regions of D-SLAM shown in FIGS. 1A-Bcould be used in an antisense approach to inhibit translation ofendogenous D-SLAM mRNA. Oligonucleotides complementary to the 5′untranslated region of the mRNA should include the complement of the AUGstart codon. Antisense oligonucleotides complementary to mRNA codingregions are less efficient inhibitors of translation but could be usedin accordance with the invention. Whether designed to hybridize to the5′-, 3′- or coding region of D-SLAM mRNA, antisense nucleic acids shouldbe at least six nucleotides in length, and are preferablyoligonucleotides ranging from 6 to about 50 nucleotides in length. Inspecific aspects the oligonucleotide is at least 10 nucleotides, atleast 17 nucleotides, at least 25 nucleotides or at least 50nucleotides.

The polynucleotides of the invention can be DNA or RNA or chimericmixtures or derivatives or modified versions thereof, single-stranded ordouble-stranded. The oligonucleotide can be modified at the base moiety,sugar moiety, or phosphate backbone, for example, to improve stabilityof the molecule, hybridization, etc. The oligonucleotide may includeother appended groups such as peptides (e.g., for targeting host cellreceptors in vivo), or agents facilitating transport across the cellmembrane (see, e.g., Letsinger et al., 1989, Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556; Lemaitre et al., 1987, Proc. Natl. Acad. Sci.84:648-652; PCT Publication No. WO88/09810, published Dec. 15, 1988) orthe blood-brain barrier (see, e.g., PCT Publication No. WO89/10134,published Apr. 25, 1988), hybridization-triggered cleavage agents. (See,e.g., Krol et al., 1988, BioTechniques 6:958-976) or intercalatingagents. (See, e.g., Zon, 1988, Pharm. Res. 5:539-549). To this end, theoligonucleotide may be conjugated to another molecule, e.g., a peptide,hybridization triggered cross-linking agent, transport agent,hybridization-triggered cleavage agent, etc.

The antisense oligonucleotide may comprise at least one modified basemoiety which is selected from the group including, but not limited to,5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl)uracil, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N-6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

The antisense oligonucleotide may also comprise at least one modifiedsugar moiety selected from the group including, but not limited to,arabinose, 2-fluoroarabinose, xylulose, and hexose.

In yet another embodiment, the antisense oligonucleotide comprises atleast one modified phosphate backbone selected from the group including,but not limited to, a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

In yet another embodiment, the antisense oligonucleotide is ana-anomeric oligonucleotide. An a-anomeric oligonucleotide forms specificdouble-stranded hybrids with complementary RNA in which, contrary to theusual b-units, the strands run parallel to each other (Gautier et al.,1987, Nucl. Acids Res. 15:6625-6641). The oligonucleotide is a2′-0-methylribonucleotide (Inoue et al., 1987, Nucl. Acids Res.15:6131-6148), or a chimeric RNA-DNA analogue (Inoue et al., 1987, FEBSLett. 215:327-330).

Polynucleotides of the invention may be synthesized by standard methodsknown in the art, e.g. by use of an automated DNA synthesizer (such asare commercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (1988, Nucl. Acids Res. 16:3209),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., 1988, Proc. Natl. Acad. Sci.U.S.A. 85:7448-7451), etc.

While antisense nucleotides complementary to the D-SLAM coding regionsequence could be used, those complementary to the transcribeduntranslated region are most preferred.

Potential antagonists according to the invention also include catalyticRNA, or a ribozyme (See, e.g., PCT International Publication WO90/11364, published Oct. 4, 1990; Sarver et al, Science 247:1222-1225(1990). While ribozymes that cleave mRNA at site specific recognitionsequences can be used to destroy D-SLAM mRNAs, the use of hammerheadribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locationsdictated by flanking regions that form complementary base pairs with thetarget mRNA. The sole requirement is that the target mRNA have thefollowing sequence of two bases: 5′-UG-3′. The construction andproduction of hammerhead ribozymes is well known in the art and isdescribed more fully in Haseloff and Gerlach, Nature 334:585-591 (1988).There are numerous potential hammerhead ribozyme cleavage sites withinthe nucleotide sequence of D-SLAM (FIGS. 1A-B). Preferably, the ribozymeis engineered so that the cleavage recognition site is located near the5′ end of the D-SLAM mRNA; i.e., to increase efficiency and minimize theintracellular accumulation of non-functional mRNA transcripts.

As in the antisense approach, the ribozymes of the invention can becomposed of modified oligonucleotides (e.g. for improved stability,targeting, etc.) and should be delivered to cells which express D-SLAMin vivo. DNA constructs encoding the ribozyme may be introduced into thecell in the same manner as described above for the introduction ofantisense encoding DNA. A preferred method of delivery involves using aDNA construct “encoding” the ribozyme under the control of a strongconstitutive promoter, such as, for example, pol III or pol II promoter,so that transfected cells will produce sufficient quantities of theribozyme to destroy endogenous D-SLAM messages and inhibit translation.Since ribozymes unlike antisense molecules, are catalytic, a lowerintracellular concentration is required for efficiency.

Antagonist/agonist compounds may be employed to inhibit the cell growthand proliferation effects of the polypeptides of the present inventionon neoplastic cells and tissues, i.e. stimulation of angiogenesis oftumors, and, therefore, retard or prevent abnormal cellular growth andproliferation, for example, in tumor formation or growth.

The antagonist/agonist may also be employed to prevent hyper-vasculardiseases, and prevent the proliferation of epithelial lens cells afterextracapsular cataract surgery. Prevention of the mitogenic activity ofthe polypeptides of the present invention may also be desirous in casessuch as restenosis after balloon angioplasty.

The antagonist/agonist may also be employed to prevent the growth ofscar tissue during wound healing.

The antagonist/agonist may also be employed to treat, diagnose, detect,and/or prevent the diseases described herein.

Binding Peptides and Other Molecules

The invention also encompasses screening methods for identifyingpolypeptides and nonpolypeptides that bind D-SLAM polypeptides, and theD-SLAM binding molecules identified thereby. These binding molecules areuseful, for example, as agonists and antagonists of D-SLAM polypeptides.Such agonists and antagonists can be used, in accordance with theinvention, in the therapeutic embodiments described in detail, below.

This method comprises the steps of:

-   -   contacting D-SLAM polypeptides or D-SLAM-like polypeptides with        a plurality of molecules; and    -   identifying a molecule that binds the D-SLAM polypeptides or        D-SLAM-like polypeptides.

The step of contacting D-SLAM polypeptides or D-SLAM-like polypeptideswith the plurality of molecules may be effected in a number of ways. Forexample, one may contemplate immobilizing D-SLAM polypeptides orD-SLAM-like polypeptides on a solid support and bringing a solution ofthe plurality of molecules in contact with the immobilized D-SLAMpolypeptides or D-SLAM-like polypeptides. Such a procedure would be akinto an affinity chromatographic process, with the affinity matrix beingcomprised of the immobilized D-SLAM polypeptides or D-SLAM-likepolypeptides. The molecules having a selective affinity for the D-SLAMpolypeptides or D-SLAM-like polypeptides can then be purified byaffinity selection. The nature of the solid support, process forattachment of the D-SLAM polypeptides or D-SLAM-like polypeptides to thesolid support, solvent, and conditions of the affinity isolation orselection are largely conventional and well known to those of ordinaryskill in the art.

Alternatively, one may also separate a plurality of polypeptides intosubstantially separate fractions comprising a subset of or individualpolypeptides. For instance, one can separate the plurality ofpolypeptides by gel electrophoresis, column chromatography, or likemethod known to those of ordinary skill for the separation ofpolypeptides. The individual polypeptides can also be produced by atransformed host cell in such a way as to be expressed on or about itsouter surface (e.g., a recombinant phage). Individual isolates can thenbe “probed” by the D-SLAM polypeptides or D-SLAM-like polypeptides,optionally in the presence of an inducer should one be required forexpression, to determine if any selective affinity interaction takesplace between the D-SLAM polypeptides or D-SLAM-like polypeptides andthe individual clone. Prior to contacting the D-SLAM polypeptides orD-SLAM-like polypeptides with each fraction comprising individualpolypeptides, the polypeptides could first be transferred to a solidsupport for additional convenience. Such a solid support may simply be apiece of filter membrane, such as one made of nitrocellulose or nylon.In this manner, positive clones could be identified from a collection oftransformed host cells of an expression library, which harbor a DNAconstruct encoding a polypeptide having a selective affinity for D-SLAMpolypeptides or D-SLAM-like polypeptides. Furthermore, the amino acidsequence of the polypeptide having a selective affinity for the D-SLAMpolypeptides or D-SLAM-like polypeptides can be determined directly byconventional means or the coding sequence of the DNA encoding thepolypeptide can frequently be determined more conveniently. The primarysequence can then be deduced from the corresponding DNA sequence. If theamino acid sequence is to be determined from the polypeptide itself, onemay use microsequencing techniques. The sequencing technique may includemass spectroscopy.

In certain situations, it may be desirable to wash away any unboundD-SLAM polypeptides or D-SLAM-like polypeptides, or alternatively,unbound polypeptides, from a mixture of the D-SLAM polypeptides orD-SLAM-like polypeptides and the plurality of polypeptides prior toattempting to determine or to detect the presence of a selectiveaffinity interaction. Such a wash step may be particularly desirablewhen the D-SLAM polypeptides or D-SLAM-like polypeptides or theplurality of polypeptides is bound to a solid support.

The plurality of molecules provided according to this method may beprovided by way of diversity libraries, such as random or combinatorialpeptide or nonpeptide libraries which can be screened for molecules thatspecifically bind D-SLAM polypeptides. Many libraries are known in theart that can be used, e.g., chemically synthesized libraries,recombinant (e.g., phage display libraries), and in vitrotranslation-based libraries. Examples of chemically synthesizedlibraries are described in Fodor et al., 1991, Science 251:767-773;Houghten et al., 1991, Nature 354:84-86; Lam et al., 1991, Nature354:82-84; Medynski, 1994, Bio/Technology 12:709-710; Gallop et al.,1994, J. Medicinal Chemistry 37(9):1233-1251; Ohlmeyer et al., 1993,Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al., 1994, Proc. Natl.Acad. Sci. USA 91:11422-11426; Houghten et al., 1992, Biotechniques13:412; Jayawickreme et al., 1994, Proc. Natl. Acad. Sci. USA91:1614-1618; Sahnon et al., 1993, Proc. Natl. Acad. Sci. USA90:11708-11712; PCT Publication No. WO 93/20242; and Brenner and Lerner,1992, Proc. Natl. Acad. Sci. USA 89:5381-5383.

Examples of phage display libraries are described in Scott and Smith,1990, Science 249:386-390; Devlin et al., 1990, Science, 249:404-406;Christian, R. B., et al., 1992, J. Mol. Biol. 227:711-718); Lenstra,1992, J. Immunol. Meth. 152:149-157; Kay et al., 1993, Gene 128:59-65;and PCT Publication No. WO 94/18318 dated Aug. 18, 1994.

In vitro translation-based libraries include but are not limited tothose described in PCT Publication No. WO 91/05058 dated Apr. 18, 1991;and Mattheakis et al., 1994, Proc. Natl. Acad. Sci. USA 91:9022-9026.

By way of examples of nonpeptide libraries, a benzodiazepine library(see e.g., Bunin et al., 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712)can be adapted for use. Peptoid libraries (Simon et al., 1992, Proc.Natl. Acad. Sci. USA 89:9367-9371) can also be used. Another example ofa library that can be used, in which the amide functionalities inpeptides have been permethylated to generate a chemically transformedcombinatorial library, is described by Ostresh et al. (1994, Proc. Natl.Acad. Sci. USA 91:11138-11142).

The variety of non-peptide libraries that are useful in the presentinvention is great. For example, Ecker and Crooke, 1995, Bio/Technology13:351-360 list benzodiazepines, hydantoins, piperazinediones,biphenyls, sugar analogs, beta-mercaptoketones, arylacetic acids,acylpiperidines, benzopyrans, cubanes, xanthines, aminimides, andoxazolones as among the chemical species that form the basis of variouslibraries.

Non-peptide libraries can be classified broadly into two types:decorated monomers and oligomers. Decorated monomer libraries employ arelatively simple scaffold structure upon which a variety functionalgroups is added. Often the scaffold will be a molecule with a knownuseful pharmacological activity. For example, the scaffold might be thebenzodiazepine structure.

Non-peptide oligomer libraries utilize a large number of monomers thatare assembled together in ways that create new shapes that depend on theorder of the monomers. Among the monomer units that have been used arecarbamates, pyrrolinones, and morpholinos. Peptoids, peptide-likeoligomers in which the side chain is attached to the alpha amino grouprather than the alpha carbon, form the basis of another version ofnon-peptide oligomer libraries. The first non-peptide oligomer librariesutilized a single type of monomer and thus contained a repeatingbackbone. Recent libraries have utilized more than one monomer, givingthe libraries added flexibility.

Screening the libraries can be accomplished by any of a variety ofcommonly known methods. See, e.g., the following references, whichdisclose screening of peptide libraries: Parmley and Smith, 1989, Adv.Exp. Med. Biol. 251:215-218; Scott and Smith, 1990, Science 249:386-390;Fowlkes et al., 1992; BioTechniques 13:422-427; Oldenburg et al., 1992,Proc. Natl. Acad. Sci. USA 89:5393-5397; Yu et al., 1994, Cell76:933-945; Staudt et al., 1988, Science 241:577-580; Bock et al., 1992,Nature 355:564-566; Tuerk et al., 1992, Proc. Natl. Acad. Sci. USA89:6988-6992; Ellington et al., 1992, Nature 355:850-852; U.S. Pat. No.5,096,815, U.S. Pat. No. 5,223,409, and U.S. Pat. No. 5,198,346, all toLadner et al.; Rebar and Pabo, 1993, Science 263:671-673; and CTPublication No. WO 94/18318.

In a specific embodiment, screening to identify a molecule that bindsD-SLAM polypeptides can be carried out by contacting the library memberswith a D-SLAM polypeptides or D-SLAM-like polypeptides immobilized on asolid phase and harvesting those library members that bind to the D-SLAMpolypeptides or D-SLAM-like polypeptides. Examples of such screeningmethods, termed “panning” techniques are described by way of example inParmley and Smith, 1988, Gene 73:305-318; Fowlkes et al., 1992,BioTechniques 13:422-427; PCT Publication No. WO 94/18318; and inreferences cited herein.

In another embodiment, the two-hybrid system for selecting interactingproteins in yeast (Fields and Song, 1989, Nature 340:245-246; Chien etal., 1991, Proc. Natl. Acad. Sci. USA 88:9578-9582) can be used toidentify molecules that specifically bind to D-SLAM polypeptides orD-SLAM-like polypeptides.

Where the D-SLAM binding molecule is a polypeptide, the polypeptide canbe conveniently selected from any peptide library, including randompeptide libraries, combinatorial peptide libraries, or biased peptidelibraries. The term “biased” is used herein to mean that the method ofgenerating the library is manipulated so as to restrict one or moreparameters that govern the diversity of the resulting collection ofmolecules, in this case peptides.

Thus, a truly random peptide library would generate a collection ofpeptides in which the probability of finding a particular amino acid ata given position of the peptide is the same for all 20 amino acids. Abias can be introduced into the library, however, by specifying, forexample, that a lysine occur every fifth amino acid or that positions 4,8, and 9 of a decapeptide library be fixed to include only arginine.Clearly, many types of biases can be contemplated, and the presentinvention is not restricted to any particular bias. Furthermore, thepresent invention contemplates specific types of peptide libraries, suchas phage displayed peptide libraries and those that utilize a DNAconstruct comprising a lambda phage vector with a DNA insert.

As mentioned above, in the case of a D-SLAM binding molecule that is apolypeptide, the polypeptide may have about 6 to less than about 60amino acid residues, preferably about 6 to about 10 amino acid residues,and most preferably, about 6 to about 22 amino acids. In anotherembodiment, a D-SLAM binding polypeptide has in the range of 15-100amino acids, or 20-50 amino acids.

The selected D-SLAM binding polypeptide can be obtained by chemicalsynthesis or recombinant expression.

Other Activities

The polypeptide of the present invention, as a result of the ability tostimulate vascular endothelial cell growth, may be employed intreatment, diagnosis, detection, and/or prevention for stimulatingre-vascularization of ischemic tissues due to various disease conditionssuch as thrombosis, arteriosclerosis, and other cardiovascularconditions. These polypeptide may also be employed to stimulateangiogenesis and limb regeneration, as discussed above.

The polypeptide may also be employed for treating, diagnosing,detecting, and/or preventing wounds due to injuries, burns,post-operative tissue repair, and ulcers since they are mitogenic tovarious cells of different origins, such as fibroblast cells andskeletal muscle cells, and therefore, facilitate the repair orreplacement of damaged or diseased tissue.

The polypeptide of the present invention may also be employed stimulateneuronal growth and to treat, diagnose, detect, and/or prevent neuronaldamage which occurs in certain neuronal disorders or neuro-degenerativeconditions such as Alzheimer's disease, Parkinson's disease, andAIDS-related complex. D-SLAM may have the ability to stimulatechondrocyte growth, therefore, they may be employed to enhance bone andperiodontal regeneration and aid in tissue transplants or bone grafts.

The polypeptide of the present invention may be also be employed toprevent skin aging due to sunburn by stimulating keratinocyte growth.

The D-SLAM polypeptide may also be employed for preventing hair loss,since FGF family members activate hair-forming cells and promotesmelanocyte growth. Along the same lines, the polypeptides of the presentinvention may be employed to stimulate growth and differentiation ofhematopoietic cells and bone marrow cells when used in combination withother cytokines.

The D-SLAM polypeptide may also be employed to maintain organs beforetransplantation or for supporting cell culture of primary tissues.

The polypeptide of the present invention may also be employed forinducing tissue of mesodermal origin to differentiate in early embryos.

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, may also increase or decrease the differentiation orproliferation of embryonic stem cells, besides, as discussed above,hematopoietic lineage.

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, may also be used to modulate mammalian characteristics, such asbody height, weight, hair color, eye color, skin, percentage of adiposetissue, pigmentation, size, and shape (e.g., cosmetic surgery).Similarly, D-SLAM polynucleotides or polypeptides, or agonists orantagonists of D-SLAM, may be used to modulate mammalian metabolismaffecting catabolism, anabolism, processing, utilization, and storage ofenergy.

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, may be used to change a mammal's mental state or physical stateby influencing biorhythms, caricadic rhythms, depression (includingdepressive disorders), tendency for violence, tolerance for pain,reproductive capabilities (preferably by Activin or Inhibin-likeactivity), hormonal or endocrine levels, appetite, libido, memory,stress, or other cognitive qualities.

D-SLAM polynucleotides or polypeptides, or agonists or antagonists ofD-SLAM, may also be used as a food additive or preservative, such as toincrease or decrease storage capabilities, fat content, lipid, protein,carbohydrate, vitamins, minerals, cofactors or other nutritionalcomponents.

The above-recited applications have uses in a wide variety of hosts.Such hosts include, but are not limited to, human, murine, rabbit, goat,guinea pig, camel, horse, mouse, rat, hamster, pig, micro-pig, chicken,goat, cow, sheep, dog, cat, non-human primate, and human. In specificembodiments, the host is a mouse, rabbit, goat, guinea pig, chicken,rat, hamster, pig, sheep, dog or cat. In preferred embodiments, the hostis a mammal. In most preferred embodiments, the host is a human.

Having generally described the invention, the same will be more readilyunderstood by reference to the following examples, which are provided byway of illustration and are not intended as limiting.

EXAMPLES Example 1 Isolation of the D-SLAM cDNA Clone from the DepositedSample

The cDNA for D-SLAM is inserted into the SalI/NotI multiple cloning siteof pCMVSport 3.0. (Life Technologies, Inc., P.O. Box 6009, Gaithersburg,Md. 20897.) pCMVSport 3.0 contains an ampicillin resistance gene and maybe transformed into E. coli strain DH10B, also available from LifeTechnologies. (See, for instance, Gruber, C. E., et al., Focus15:59-(1993).)

Two approaches can be used to isolate D-SLAM from the deposited sample.First, a specific polynucleotide of SEQ ID NO:1 with 30-40 nucleotidesis synthesized using an Applied Biosystems DNA synthesizer according tothe sequence reported. The oligonucleotide is labeled, for instance,with ³²P-γ-ATP using T4 polynucleotide kinase and purified according toroutine methods. (E.g., Maniatis et al., Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Press, Cold Spring, N.Y. (1982).) The plasmidmixture is transformed into a suitable host (such as XL-1 Blue(Stratagene)) using techniques known to those of skill in the art, suchas those provided by the vector supplier or in related publications orpatents. The transformants are plated on 1.5% agar plates (containingthe appropriate selection agent, e.g., ampicillin) to a density of about150 transformants (colonies) per plate. These plates are screened usingNylon membranes according to routine methods for bacterial colonyscreening (e.g., Sambrook et al., Molecular Cloning: A LaboratoryManual, 2nd Edit., (1989), Cold Spring Harbor Laboratory Press, pages1.93 to 1.104), or other techniques known to those of skill in the art.

Alternatively, two primers of 17-20 nucleotides derived from both endsof the SEQ ID NO:1 (i.e., within the region of SEQ ID NO:1 bounded bythe 5′ NT and the 3′ NT of the clone) are synthesized and used toamplify the D-SLAM cDNA using the deposited cDNA plasmid as a template.The polymerase chain reaction is carried out under routine conditions,for instance, in 25 μl of reaction mixture with 0.5 ug of the above cDNAtemplate. A convenient reaction mixture is 1.5-5 mM MgCl₂, 0.01% (w/v)gelatin, 20 μM each of dATP, dCTP, dGTP, dTTP, 25 pmol of each primerand 0.25 Unit of Taq polymerase. Thirty five cycles of PCR (denaturationat 94° C. for 1 min; annealing at 55° C. for 1 min; elongation at 72° C.for 1 min) are performed with a Perkin-Elmer Cetus automated thermalcycler. The amplified product is analyzed by agarose gel electrophoresisand the DNA band with expected molecular weight is excised and purified.The PCR product is verified to be the selected sequence by subcloningand sequencing the DNA product.

Several methods are available for the identification of the 5′ or 3′non-coding portions of the D-SLAM gene which may not be present in thedeposited clone. These methods include but are not limited to, filterprobing, clone enrichment using specific probes, and protocols similaror identical to 5′ and 3′ “RACE” protocols which are well known in theart. For instance, a method similar to 5′ RACE is available forgenerating the missing 5′ end of a desired full-length transcript.(Fromont-Racine et al., Nucleic Acids Res. 21(7):1683-1684 (1993).)

Briefly, a specific RNA oligonucleotide is ligated to the 5′ ends of apopulation of RNA presumably containing full-length gene RNAtranscripts. A primer set containing a primer specific to the ligatedRNA oligonucleotide and a primer specific to a known sequence of theD-SLAM gene of interest is used to PCR amplify the 5′ portion of theD-SLAM full-length gene. This amplified product may then be sequencedand used to generate the full length gene.

This above method starts with total RNA isolated from the desiredsource, although poly-A+ RNA can be used. The RNA preparation can thenbe treated with phosphatase if necessary to eliminate 5′ phosphategroups on degraded or damaged RNA which may interfere with the later RNAligase step. The phosphatase should then be inactivated and the RNAtreated with tobacco acid pyrophosphatase in order to remove the capstructure present at the 5′ ends of messenger RNAs. This reaction leavesa 5′ phosphate group at the 5′ end of the cap cleaved RNA which can thenbe ligated to an RNA oligonucleotide using T4 RNA ligase.

This modified RNA preparation is used as a template for first strandcDNA synthesis using a gene specific oligonucleotide. The first strandsynthesis reaction is used as a template for PCR amplification of thedesired 5′ end using a primer specific to the ligated RNAoligonucleotide and a primer specific to the known sequence of the geneof interest. The resultant product is then sequenced and analyzed toconfirm that the 5′ end sequence belongs to the D-SLAM gene.

Example 2 Isolation of D-SLAM Genomic Clones

A human genomic P1 library (Genomic Systems, Inc.) is screened by PCRusing primers selected for the cDNA sequence corresponding to SEQ IDNO:1., according to the method described in Example 1. (See also,Sambrook.)

Example 3 Tissue Distribution of D-SLAM Polypeptides

Tissue distribution of mRNA expression of D-SLAM is determined usingprotocols for Northern blot analysis, described by, among others,Sambrook et al. For example, a D-SLAM probe produced by the methoddescribed in Example 1 is labeled with P³² using the rediprime™ DNAlabeling system (Amersham Life Science), according to manufacturer'sinstructions. After labeling, the probe is purified using CHROMASPIN-100™ column (Clontech Laboratories, Inc.), according tomanufacturer's protocol number PT1200-1. The purified labeled probe isthen used to examine various human tissues for mRNA expression.

Multiple Tissue Northern (MTN) blots containing various human tissues(H) or human immune system tissues (IM) (Clontech) are examined with thelabeled probe using ExpressHyb™ hybridization solution (Clontech)according to manufacturer's protocol number PT1190-1. Followinghybridization and washing, the blots are mounted and exposed to film at−70° C. overnight, and the films developed according to standardprocedures.

Example 4 Chromosomal Mapping of D-SLAM

An oligonucleotide primer set is designed according to the sequence atthe 5′ end of SEQ ID NO:1. This primer preferably spans about 100nucleotides. This primer set is then used in a polymerase chain reactionunder the following set of conditions: 30 seconds, 95° C.; 1 minute, 56°C.; 1 minute, 70° C. This cycle is repeated 32 times followed by one 5minute cycle at 70° C. Human, mouse, and hamster DNA is used as templatein addition to a somatic cell hybrid panel containing individualchromosomes or chromosome fragments (Bios, Inc). The reactions isanalyzed on either 8% polyacrylamide gels or 3.5% agarose gels.Chromosome mapping is determined by the presence of an approximately 100bp PCR fragment in the particular somatic cell hybrid.

Example 5 Bacterial Expression of D-SLAM

D-SLAM polynucleotide encoding a D-SLAM polypeptide invention isamplified using PCR oligonucleotide primers corresponding to the 5′ and3′ ends of the DNA sequence, as outlined in Example 1, to synthesizeinsertion fragments. The primers used to amplify the cDNA insert shouldpreferably contain restriction sites, such as BamHI and XbaI, at the 5′end of the primers in order to clone the amplified product into theexpression vector. For example, BamHI and XbaI correspond to therestriction enzyme sites on the bacterial expression vector pQE-9.(Qiagen, Inc., Chatsworth, Calif.). This plasmid vector encodesantibiotic resistance (Ampr), a bacterial origin of replication (ori),an IPTG-regulatable promoter/operator (P/O), a ribosome binding site(RBS), a 6-histidine tag (6-His), and restriction enzyme cloning sites.

The pQE-9 vector is digested with BamHI and XbaI and the amplifiedfragment is ligated into the pQE-9 vector maintaining the reading frameinitiated at the bacterial RBS. The ligation mixture is then used totransform the E. coli strain M15/rep4 (Qiagen, Inc.) which containsmultiple copies of the plasmid pREP4, which expresses the lacI repressorand also confers kanamycin resistance (Kan^(r)). Transformants areidentified by their ability to grow on LB plates andampicillin/kanamycin resistant colonies are selected. Plasmid DNA isisolated and confirmed by restriction analysis.

Alternatively, a construct containing DNA encoding amino acid Q24-D233of SEQ ID NO:2 can be inserted into pQE70. This construct places a HIStag (6 histidines) at the C-terminus of the predicted extracellulardomain of D-SLAM. Primers that can be used include a 5′ primercontaining a Sph restriction site, shown in bold:GCAGCAGCATGCAAGTGCTGAGCAAAGTCGGGGGCTCGGTGCTG (SEQ ID NO: 14) and a 3′primer, containing a BglII restriction site, shown in bold:GCAGCAAGATCTATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO: 15). Thisconstruct uses an ATG as a start codon contained within the SphI site,then reading into Q24 of SEQ ID NO:2, and continues until D233 of SEQ IDNO:2. The amino acid sequence encoded by this construct is as follows:MQVLSKVGGSVLLVAARPPGFQVREAIWRSLWPSEE (SEQ ID NO:16)LLATFFRGSLETLYHSRFLGRAQLHSNLSLELGPLESGDSGNFSVLMVDTRGQPWTQTLQLKVYDAVPRPVVQVFIAVERDAQPSKTCQVFLSCWAPNISEITYSWRRETTMDFGMEPHSLFTDGQVLSISLGPGDRDVAYSCIVSNPVSWDLATVTPWDSCHHEAAPGKASYKDQVLSKVGGSVLLVAARPPGFQVREAIWRSLWPSEELLATFFRGSLETLYHSRFLGRAQLHSNLSLELGPLESGDSGNFSVLMVDTRGQPWTQTLQLKVYDAVPRPVVQVFIAVERDAQPSKTCQVFLSCWAPNISEITYSWRRETTMDFGMEPHSLFTDGQVLSISLGPGDRDVAYSCIVSNPVS WDLATVTPWDSCHHEAAPGKASYKDHHHHHH.

Alternatively, a His tag can be placed on the N-terminus of thepredicted mature form containing only the extracellular domain of D-SLAM(e.g., corresponding to A23-D233 of SEQ ID NO:2). In this example, the5′ primer, containing a BamHI restriction site, indicated in bold, canbe used: GCAGCAGGATCCGCCCAAGTGCTGAGCAAAGTCGGGGGCTCGGTG (SEQ ID NO: 17)and a 3′ primer, containing a HindIII restriction site, indicated inbold, can be used: GCAGCAAAGCTTTTAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQID NO:18). These primer can be used to amplify DNA encoding A23-D233,and then the generated product can be inserted into pQE9. This constructputs a His tag on the N-terminus of the predicted mature extracellulardomain of D-SLAM. The His tag will be followed by the Gly-Ser of theBamHI site, and this will then be followed by A23 of SEQ ID NO:2. Thisconstruct will continue through D233 of SEQ ID NO:2, and will befollowed by a TAA stop codon. The amino acid sequence encoded by thisconstruct is as follows: MRGSHHHHHHGSAQVLSKVGGSVLLVAARPPGFQVR (SEQ IDNO:19) EAIWRSLWPSEELLATFFRGSLETLYHSRFLGRAQLHSNLSLELGPLESGDSGNFSVLMVDTRGQPWTQTLQLKVYDAVPRPVVQVFIAVERDAQPSKTCQVFLSCWAPNISEITYSWRRETTMDFGMEPHSLFTDGQVLSISLGPGDRDVAYSCIVSNPVSWDLATVTPWDSCHHEAAP GKASYKD.

Additionally, a mature form containing only the extracellular domain ofD-SLAM (amino acids A23 to D233 of SEQ ID NO:2) can also be insertedinto an E. coli expression vector, such as pHE4 (see below). In thisexample, the 5′ primer, containing a Nde restriction site, indicated inbold, can be used: GCAGCACATATGGCCCAAGTGCTGAGCAAAGTCG (SEQ ID NO: 20)and a 3′ primer containing an Asp718 restriction site, shown in bold,can be used: GCAGCAGGTACCTTACTAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ IDNO: 21).

Clones containing the desired constructs are grown overnight (O/N) inliquid culture in LB media supplemented with both Amp (100 ug/ml) andKan (25 ug/ml). The O/N culture is used to inoculate a large culture ata ratio of 1:100 to 1:250. The cells are grown to an optical density 600(O.D.⁶⁰⁰) of between 0.4 and 0.6. IPTG(Isopropyl-B-D-thiogalactopyranoside) is then added to a finalconcentration of 1 mM. IPTG induces by inactivating the lacI repressor,clearing the P/O leading to increased gene expression.

Cells are grown for an extra 3 to 4 hours. Cells are then harvested bycentrifugation (20 mins at 6000×g). The cell pellet is solubilized inthe chaotropic agent 6 Molar Guanidine HCl by stirring for 3-4 hours at4° C. The cell debris is removed by centrifugation, and the supernatantcontaining the polypeptide is loaded onto a nickel-nitrilo-tri-aceticacid (“Ni-NTA”) affinity resin column (available from QIAGEN, Inc.,supra). Proteins with a 6×His tag bind to the Ni-NTA resin with highaffinity and can be purified in a simple one-step procedure (for detailssee: The QIAexpressionist (1995) QIAGEN, Inc., supra).

Briefly, the supernatant is loaded onto the column in 6 M guanidine-HCl,pH 8, the column is first washed with 10 volumes of 6 M guanidine-HCl,pH 8, then washed with 10 volumes of 6 M guanidine-HCl pH 6, and finallythe polypeptide is eluted with 6 M guanidine-HCl, pH 5.

The purified D-SLAM protein is then renatured by dialyzing it againstphosphate-buffered saline (PBS) or 50 mM Na-acetate, pH 6 buffer plus200 mM NaCl. Alternatively, the D-SLAM protein can be successfullyrefolded while immobilized on the Ni-NTA column. The recommendedconditions are as follows: renature using a linear 6M-1M urea gradientin 500 mM NaCl, 20% glycerol, 20 mM Tris/HCl pH 7.4, containing proteaseinhibitors. The renaturation should be performed over a period of 1.5hours or more. After renaturation the proteins are eluted by theaddition of 250 mM immidazole. Immidazole is removed by a finaldialyzing step against PBS or 50 mM sodium acetate pH 6 buffer plus 200mM NaCl. The purified D-SLAM protein is stored at 4° C. or frozen at−80° C.

In addition to the above expression vector, the present inventionfurther includes an expression vector comprising phage operator andpromoter elements operatively linked to a D-SLAM polynucleotide, calledpHE4a. (ATCC Accession Number 209645, deposited Feb. 25, 1998.) Thisvector contains: 1) a neomycinphosphotransferase gene as a selectionmarker, 2) an E. coli origin of replication, 3) a T5 phage promotersequence, 4) two lac operator sequences, 5) a Shine-Delgarno sequence,and 6) the lactose operon repressor gene (lacIq). The origin ofreplication (oriC) is derived from pUC19 (LTI, Gaithersburg, Md.). Thepromoter sequence and operator sequences are made synthetically.

DNA can be inserted into the pHEa by restricting the vector with NdeIand XbaI, BamHI, XhoI, or Asp718, running the restricted product on agel, and isolating the larger fragment (the stuffer fragment should beabout 310 base pairs). The DNA insert is generated according to the PCRprotocol described in Example 1, using PCR primers having restrictionsites for NdeI (5′ primer) and XbaI, BamHI, XhoI, or Asp718 (3′ primer).The PCR insert is gel purified and restricted with compatible enzymes.The insert and vector are ligated according to standard protocols.

The engineered vector could easily be substituted in the above protocolto express protein in a bacterial system.

Example 6 Purification of D-SLAM Polypeptide from an Inclusion Body

The following alternative method can be used to purify D-SLAMpolypeptide expressed in E. coli when it is present in the form ofinclusion bodies. Unless otherwise specified, all of the following stepsare conducted at 4-10° C.

Upon completion of the production phase of the E. coli fermentation, thecell culture is cooled to 4-10° C. and the cells harvested by continuouscentrifugation at 15,000 rpm (Heraeus Sepatech). On the basis of theexpected yield of protein per unit weight of cell paste and the amountof purified protein required, an appropriate amount of cell paste, byweight, is suspended in a buffer solution containing 100 mM Tris, 50 mMEDTA, pH 7.4. The cells are dispersed to a homogeneous suspension usinga high shear mixer.

The cells are then lysed by passing the solution through amicrofluidizer (Microfuidics, Corp. or APV Gaulin, Inc.) twice at4000-6000 psi. The homogenate is then mixed with NaCl solution to afinal concentration of 0.5 M NaCl, followed by centrifugation at 7000×gfor 15 min. The resultant pellet is washed again using 0.5M NaCl, 100 mMTris, 50 mM EDTA, pH 7.4.

The resulting washed inclusion bodies are solubilized with 1.5 Mguanidine hydrochloride (GuHCl) for 2-4 hours. After 7000×gcentrifugation for 15 min., the pellet is discarded and the polypeptidecontaining supernatant is incubated at 4° C. overnight to allow furtherGuHCl extraction.

Following high speed centrifugation (30,000×g) to remove insolubleparticles, the GuHCl solubilized protein is refolded by quickly mixingthe GuHCl extract with 20 volumes of buffer containing 50 mM sodium, pH4.5, 150 mM NaCl, 2 mM EDTA by vigorous stirring. The refolded dilutedprotein solution is kept at 4° C. without mixing for 12 hours prior tofurther purification steps.

To clarify the refolded polypeptide solution, a previously preparedtangential filtration unit equipped with 0.16 um membrane filter withappropriate surface area (e.g., Filtron), equilibrated with 40 mM sodiumacetate, pH 6.0 is employed. The filtered sample is loaded onto a cationexchange resin (e.g., Poros HS-50, Perseptive Biosystems). The column iswashed with 40 mM sodium acetate, pH 6.0 and eluted with 250 mM, 500 mM,1000 mM, and 1500 mM NaCl in the same buffer, in a stepwise manner. Theabsorbance at 280 nm of the effluent is continuously monitored.Fractions are collected and further analyzed by SDS-PAGE.

Fractions containing the D-SLAM polypeptide are then pooled and mixedwith 4 volumes of water. The diluted sample is then loaded onto apreviously prepared set of tandem columns of strong anion (Poros HQ-50,Perseptive Biosystems) and weak anion (Poros CM-20, PerseptiveBiosystems) exchange resins. The columns are equilibrated with 40 mMsodium acetate, pH 6.0. Both columns are washed with 40 mM sodiumacetate, pH 6.0, 200 mM NaCl. The CM-20 column is then eluted using a 10column volume linear gradient ranging from 0.2 M NaCl, 50 mM sodiumacetate, pH 6.0 to 1.0 M NaCl, 50 mM sodium acetate, pH 6.5. Fractionsare collected under constant A₂₈₀ monitoring of the effluent. Fractionscontaining the polypeptide (determined, for instance, by 16% SDS-PAGE)are then pooled.

The resultant D-SLAM polypeptide should exhibit greater than 95% purityafter the above refolding and purification steps. No major contaminantbands should be observed from Commassie blue stained 16% SDS-PAGE gelwhen 5 ug of purified protein is loaded. The purified D-SLAM protein canalso be tested for endotoxin/LPS contamination, and typically the LPScontent is less than 0.1 ng/ml according to LAL assays.

Example 7 Cloning and Expression of D-SLAM in a Baculovirus ExpressionSystem

In this example, the plasmid shuttle vector pA2 is used to insert D-SLAMpolynucleotide into a baculovirus to express D-SLAM. This expressionvector contains the strong polyhedrin promoter of the Autographacalifornica nuclear polyhedrosis virus (AcMNPV) followed by convenientrestriction sites such as BamHI, Xba I and Asp718. The polyadenylationsite of the simian virus 40 (“SV40”) is used for efficientpolyadenylation. For easy selection of recombinant virus, the plasmidcontains the beta-galactosidase gene from E. coli under control of aweak Drosophila promoter in the same orientation, followed by thepolyadenylation signal of the polyhedrin gene. The inserted genes areflanked on both sides by viral sequences for cell-mediated homologousrecombination with wild-type viral DNA to generate a viable virus thatexpress the cloned D-SLAM polynucleotide.

Many other baculovirus vectors can be used in place of the vector above,such as pAc373, pVL941, and pAcIM1, as one skilled in the art wouldreadily appreciate, as long as the construct provides appropriatelylocated signals for transcription, translation, secretion and the like,including a signal peptide and an in-frame AUG as required. Such vectorsare described, for instance, in Luckow et al., Virology 170:31-39(1989).

Specifically, the D-SLAM cDNA sequence contained in the deposited clone,including the AUG initiation codon and any naturally associated leadersequence, is amplified using the PCR protocol described in Example 1. Ifthe naturally occurring signal sequence is used to produce the secretedprotein, the pA2 vector does not need a second signal peptide.Alternatively, the vector can be modified (pA2 GP) to include abaculovirus leader sequence, using the standard methods described inSummers et al., “A Manual of Methods for Baculovirus Vectors and InsectCell Culture Procedures,” Texas Agricultural Experimental StationBulletin No. 1555 (1987).

Fragments of D-SLAM can be expressed from the baculovirus system. Forexample, the predicted extracellular domain (M1-K232 of SEQ ID NO:2) canbe inserted into pA2 using the primers described throughout the Examplesection.

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel.

The plasmid is digested with the corresponding restriction enzymes andoptionally, can be dephosphorylated using calf intestinal phosphatase,using routine procedures known in the art. The DNA is then isolated froma 1% agarose gel using a commercially available kit (“Geneclean” BIO 101Inc., La Jolla, Calif.).

The fragment and the dephosphorylated plasmid are ligated together withT4 DNA ligase. E. coli HB101 or other suitable E. coli hosts such asXL-1 Blue (Stratagene Cloning Systems, La Jolla, Calif.) cells aretransformed with the ligation mixture and spread on culture plates.Bacteria containing the plasmid are identified by digesting DNA fromindividual colonies and analyzing the digestion product by gelelectrophoresis. The sequence of the cloned fragment is confirmed by DNAsequencing.

Five ug of a plasmid containing the polynucleotide is co-transfectedwith 1.0 ug of a commercially available linearized baculovirus DNA(“BaculoGold™ baculovirus DNA”, Pharmingen, San Diego, Calif.), usingthe lipofection method described by Felgner et al., Proc. Natl. Acad.Sci. USA 84:7413-7417 (1987). One ug of BaculoGold™ virus DNA and 5 ugof the plasmid are mixed in a sterile well of a microtiter platecontaining 50 ul of serum-free Grace's medium (Life Technologies Inc.,Gaithersburg, Md.). Afterwards, 10 ul Lipofectin plus 90 ul Grace'smedium are added, mixed and incubated for 15 minutes at roomtemperature. Then the transfection mixture is added drop-wise to Sf9insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with1 ml Grace's medium without serum. The plate is then incubated for 5hours at 27 degrees C. The transfection solution is then removed fromthe plate and 1 ml of Grace's insect medium supplemented with 10% fetalcalf serum is added. Cultivation is then continued at 27 degrees C. forfour days.

After four days the supernatant is collected and a plaque assay isperformed, as described by Summers and Smith, supra. An agarose gel with“Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easyidentification and isolation of gal-expressing clones, which produceblue-stained plaques. (A detailed description of a “plaque assay” ofthis type can also be found in the user's guide for insect cell cultureand baculovirology distributed by Life Technologies Inc., Gaithersburg,page 9-10.) After appropriate incubation, blue stained plaques arepicked with the tip of a micropipettor (e.g., Eppendorf). The agarcontaining the recombinant viruses is then resuspended in amicrocentrifuge tube containing 200 ul of Grace's medium and thesuspension containing the recombinant baculovirus is used to infect Sf9cells seeded in 35 mm dishes. Four days later the supernatants of theseculture dishes are harvested and then they are stored at 4° C.

To verify the expression of the polypeptide, Sf9 cells are grown inGrace's medium supplemented with 10% heat-inactivated FBS. The cells areinfected with the recombinant baculovirus containing the polynucleotideat a multiplicity of infection (“MOI”) of about 2. If radiolabeledproteins are desired, 6 hours later the medium is removed and isreplaced with SF900 II medium minus methionine and cysteine (availablefrom Life Technologies Inc., Rockville, Md.). After 42 hours, 5 uCi of³⁵S-methionine and 5 uCi ³⁵S-cysteine (available from Amersham) areadded. The cells are further incubated for 16 hours and then areharvested by centrifugation. The proteins in the supernatant as well asthe intracellular proteins are analyzed by SDS-PAGE followed byautoradiography (if radiolabeled).

Microsequencing of the amino acid sequence of the amino terminus ofpurified protein may be used to determine the amino terminal sequence ofthe produced D-SLAM protein.

Example 8 Expression of D-SLAM in Mammalian Cells

D-SLAM polypeptide can be expressed in a mammalian cell. A typicalmammalian expression vector contains a promoter element, which mediatesthe initiation of transcription of mRNA, a protein coding sequence, andsignals required for the termination of transcription andpolyadenylation of the transcript. Additional elements includeenhancers, Kozak sequences and intervening sequences flanked by donorand acceptor sites for RNA splicing. Highly efficient transcription isachieved with the early and late promoters from SV40, the long terminalrepeats (LTRs) from Retroviruses, e.g., RSV, HTLVI, HIVI and the earlypromoter of the cytomegalovirus (CMV). However, cellular elements canalso be used (e.g., the human actin promoter).

Suitable expression vectors for use in practicing the present inventioninclude, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala,Sweden), pRSVcat (ATCC 37152), pSV2DHFR (ATCC 37146), pBC12MI (ATCC67109), pCMVSport 2.0, and pCMVSport 3.0. Mammalian host cells thatcould be used include, human Hela, 293, H9 and Jurkat cells, mouseNIH3T3 and C127 cells, Cos 1, Cos 7 and CV1, quail QC1-3 cells, mouse Lcells and Chinese hamster ovary (CHO) cells.

Alternatively, D-SLAM polypeptide can be expressed in stable cell linescontaining the D-SLAM polynucleotide integrated into a chromosome. Theco-transfection with a selectable marker such as DHFR, gpt, neomycin,hygromycin allows the identification and isolation of the transfectedcells.

The transfected D-SLAM gene can also be amplified to express largeamounts of the encoded protein. The DHFR (dihydrofolate reductase)marker is useful in developing cell lines that carry several hundred oreven several thousand copies of the gene of interest. (See, e.g., Alt,F. W., et al., J. Biol. Chem. 253:1357-1370 (1978); Hamlin, J. L. andMa, C., Biochem. et Biophys. Acta, 1097:107-143 (1990); Page, M. J. andSydenham, M. A., Biotechnology 9:64-68 (1991).) Another useful selectionmarker is the enzyme glutamine synthase (GS) (Murphy et al., Biochem J.227:277-279 (1991); Bebbington et al., Bio/Technology 10:169-175 (1992).Using these markers, the mammalian cells are grown in selective mediumand the cells with the highest resistance are selected. These cell linescontain the amplified gene(s) integrated into a chromosome. Chinesehamster ovary (CHO) and NSO cells are often used for the production ofproteins.

Vectors which use glutamine synthase (GS) or DHFR as the selectablemarkers can be amplified in the presence of the drugs methioninesulphoximine or methotrexate, respectively. An advantage of glutaminesynthase based vectors are the availabilty of cell lines (e.g., themurine myeloma cell line, NS0) which are glutamine synthase negative. Itis also possible to amplify vectors that utilize glutamine synthaseselection in glutamine synthase expressing cells (e.g., Chinese HamsterOvary (CHO) cells), however, by providing additional inhibitor toprevent the functioning of the endogenous gene. A glutamine synthaseexpression system and components thereof are detailed in PCTpublications: WO87/04462; WO86/05807; WO89/01036; WO89/10404; andWO91/06657 which are hereby incorporated in their entireties byreference herein. Additionally, glutamine synthase expression vectorscan be obtained from Lonza Biologics, Inc. (Portsmouth, N.H.).Expression and production of monoclonal antibodies using a GS expressionsystem in murine myeloma cells is described in Bebbington et al.,Bio/technology 10:169(1992) and in Biblia and Robinson Biotechnol. Prog.11:1 (1995) which are herein incorporated by reference.

Derivatives of the plasmid pSV2-DHFR (ATCC Accession No. 37146), theexpression vectors pC4 (ATCC Accession No. 209646) and pC6 (ATCCAccession No.209647) contain the strong promoter (LTR) of the RousSarcoma Virus (Cullen et al., Molecular and Cellular Biology, 438-447(March, 1985)) plus a fragment of the CMV-enhancer (Boshart et al., Cell41:521-530 (1985).) Multiple cloning sites, e.g., with the restrictionenzyme cleavage sites BamHI, XbaI and Asp718, facilitate the cloning ofD-SLAM. The vectors also contain the 3′ intron, the polyadenylation andtermination signal of the rat preproinsulin gene, and the mouse DHFRgene under control of the SV40 early promoter.

Specifically, the plasmid pC6 or pC4 is digested appropriate restrictionenzymes and then dephosphorylated using calf intestinal phosphates byprocedures known in the art. The vector is then isolated from a 1%agarose gel.

D-SLAM polynucleotide is amplified according to the protocol outlined inExample 1. If a naturally occurring signal sequence is used to produce asecreted protein, the vector does not need a second signal peptide.Alternatively, if a naturally occurring signal sequence is not used, thevector can be modified to include a heterologous signal sequence in aneffort to secrete the protein from the cell. (See, e.g., WO 96/34891.)

Specifically, the full length D-SLAM protein can be expressed from amammalian vector, such as pC4, using the following primers: The 5′primer, containing a BamHI in bold, is as follows:GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ ID NO:22) whilethe 3′ primer contains a Xba site shown in bold:GCAGCATCTAGATTATGGCAGATCCTGCACAAGGGGGTTCTCTGTC (SEQ ID NO: 23). Thisconstruct should produce a transmembrane protein that will be expressedon the external cell surface.

Alternatively, a construct containing only the soluble portion of D-SLAMcan be made by inserting the predicted extracellular domain of D-SLAM inpC4. For example, DNA encoding M1-K232 of SEQ ID NO:2 can be in insertedinto pC4, using a 5′ primer, containing a BamHI restriction site shownin bold: GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ ID NO:22) and a 3′ primer, containing a Xba restriction site shown in bold:GCAGCATCTAGATTATTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO: 24).

The amplified fragment is isolated from a 1% agarose gel using acommercially available kit (“Geneclean,” BIO 101 Inc., La Jolla,Calif.). The fragment then is digested with appropriate restrictionenzymes and again purified on a 1% agarose gel. The amplified fragmentis then digested with the same restriction enzyme and purified on a 1%agarose gel. The isolated fragment and the dephosphorylated vector arethen ligated with T4 DNA ligase. E. coli HB101 or XL-1 Blue cells arethen transformed and bacteria are identified that contain the fragmentinserted into plasmid pC6 or pC4 using, for instance, restriction enzymeanalysis.

Chinese hamster ovary cells lacking an active DHFR gene is used fortransfection. Five μg of the expression plasmid pC6 or pC4 iscotransfected with 0.5 ug of the plasmid pSVneo using lipofectin(Felgner et al., supra). The plasmid pSV2-neo contains a dominantselectable marker, the neo gene from Tn5 encoding an enzyme that confersresistance to a group of antibiotics including G418. The cells areseeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days,the cells are trypsinized and seeded in hybridoma cloning plates(Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50ng/ml of metothrexate plus 1 mg/ml G418. After about 10-14 days singleclones are trypsinized and then seeded in 6-well petri dishes or 10 mlflasks using different concentrations of methotrexate (50 nM, 100 nM,200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations ofmethotrexate are then transferred to new 6-well plates containing evenhigher concentrations of methotrexate (1 uM, 2 uM, 5 uM, 10 mM, 20 mM).The same procedure is repeated until clones are obtained which grow at aconcentration of 100-200 uM. Expression of D-SLAM is analyzed, forinstance, by SDS-PAGE and Western blot or by reversed phase HPLCanalysis.

Example 9 Construction of N-Terminal and/or C-Terminal Deletion Mutants

The following general approach may be used to clone a N-terminal orC-terminal deletion D-SLAM deletion mutant. Generally, twooligonucleotide primers of about 15-25 nucleotides are derived from thedesired 5′ and 3′ positions of a polynucleotide of SEQ ID NO:1. The 5′and 3′ positions of the primers are determined based on the desiredD-SLAM polynucleotide fragment. An initiation and stop codon are addedto the 5′ and 3′ primers respectively, if necessary, to express theD-SLAM polypeptide fragment encoded by the polynucleotide fragment.Preferred D-SLAM polynucleotide fragments are those encoding theN-terminal and C-terminal deletion mutants disclosed above in the“Polynucleotide and Polypeptide Fragments” section of the Specification.

Additional nucleotides containing restriction sites to facilitatecloning of the D-SLAM polynucleotide fragment in a desired vector mayalso be added to the 5′ and 3′ primer sequences. The D-SLAMpolynucleotide fragment is amplified from genomic DNA or from thedeposited cDNA clone using the appropriate PCR oligonucleotide primersand conditions discussed herein or known in the art. The D-SLAMpolypeptide fragments encoded by the D-SLAM polynucleotide fragments ofthe present invention may be expressed and purified in the same generalmanner as the full length polypeptides, although routine modificationsmay be necessary due to the differences in chemical and physicalproperties between a particular fragment and full length polypeptide.

As a means of exemplifying but not limiting the present invention, thepolynucleotide encoding the D-SLAM polypeptide fragment Leu-35 toThr-276 is amplified and cloned as follows: A 5′ primer is generatedcomprising a restriction enzyme site followed by an initiation codon inframe with the polynucleotide sequence encoding the N-terminal portionof the polypeptide fragment beginning with Leu-35. A complementary 3′primer is generated comprising a restriction enzyme site followed by astop codon in frame with the polynucleotide sequence encoding C-terminalportion of the D-SLAM polypeptide fragment ending with Thr-276.

The amplified polynucleotide fragment and the expression vector aredigested with restriction enzymes which recognize the sites in theprimers. The digested polynucleotides are then ligated together. TheD-SLAM polynucleotide fragment is inserted into the restrictedexpression vector, preferably in a manner which places the D-SLAMpolypeptide fragment coding region downstream from the promoter. Theligation mixture is transformed into competent E. coli cells usingstandard procedures and as described in the Examples herein. Plasmid DNAis isolated from resistant colonies and the identity of the cloned DNAconfirmed by restriction analysis, PCR and DNA sequencing.

Example 10 Protein Fusions of D-SLAM

D-SLAM polypeptides are preferably fused to other proteins. These fusionproteins can be used for a variety of applications. For example, fusionof D-SLAM polypeptides to His-tag, HA-tag, protein A, IgG domains, andmaltose binding protein facilitates purification. (See Example 5; seealso EP A 394,827; Traunecker, et al., Nature 331:84-86 (1988).)Similarly, fusion to IgG-1, IgG-3, and albumin increases the halflifetime in vivo. Nuclear localization signals fused to D-SLAM polypeptidescan target the protein to a specific subcellular localization, whilecovalent heterodimer or homodimers can increase or decrease the activityof a fusion protein. Fusion proteins can also create chimeric moleculeshaving more than one function. Finally, fusion proteins can increasesolubility and/or stability of the fused protein compared to thenon-fused protein. All of the types of fusion proteins described abovecan be made by modifying the following protocol, which outlines thefusion of a polypeptide to an IgG molecule, or the protocol described inExample 5.

Briefly, the human Fc portion of the IgG molecule can be PCR amplified,using primers that span the 5′ and 3′ ends of the sequence describedbelow. These primers also should have convenient restriction enzymesites that will facilitate cloning into an expression vector, preferablya mammalian expression vector.

For example, if pC4 (Accession No. 209646) is used, the human Fc portioncan be ligated into the BamHI cloning site. Note that the 3′ BamHI siteshould be destroyed. Next, the vector containing the human Fc portion isre-restricted with BamHI, linearizing the vector, and D-SLAMpolynucleotide, isolated by the PCR protocol described in Example 1, isligated into this BamHI site. Note that the polynucleotide is clonedwithout a stop codon, otherwise a fusion protein will not be produced.

Examples of primers that can be used to amplify D-SLAM polypeptides,such as the predicted extracellular domain of D-SLAM include: a 5′primer containing a BamHI restriction site shown in bold:GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ ID NO: 22) anda 3′ primer, containing a Xba restriction site shown in bold:GCAGCATCTAGAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO: 25). Usingthese primers, the construct will express the predicted extracellulardomain of D-SLAM fused to Fc in pC4.

If the naturally occurring signal sequence is used to produce thesecreted protein, pC4 does not need a second signal peptide.Alternatively, if the naturally occurring signal sequence is not used,the vector can be modified to include a heterologous signal sequence.(See, e.g., WO 96/34891.)

Human IgG Fc region: GGGATCCGGAGCCCAAATCTTCTGACAAAACTCACAC (SEQ ID NO:4)ATGCCCACCGTGCCCAGCACCTGAATTCGAGGGTGCACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACTCCTGAGGTCACATGCGTGGTGGTGGACGTAAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAACCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCAAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAA ATGAGTGCGACGGCCGCGACTCTAGAGGAT

Alternatively, this same region can be inserted in pA2, creating afusion of Fc with the predicted extracellular domain of D-SLAM. Examplesof primer that can be used to amplify the predicted extracellular domainof D-SLAM include: a 5′ primer containing a BamHI restriction site shownin bold: GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ ID NO:22) and a 3′ primer, containing a Xba restriction site shown in bold:GCAGCATCTAGAATCTTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO: 25). Thisconstruct will express the predicted extracellular domain of D-SLAMfused to Fc in pA2.

These two above constructs were expressed in their appropriate hostcells (e.g., pC4 in a mammalian system, while pA2 in a baculovirussystem), both systems produced a truncated protein, lacking thepredicted signal sequence and beginning with A23 of SEQ ID NO:2. Thus,as was predicted, both baculovirus and mammalian systems process D-SLAMto amino acid A23 of SEQ ID NO:2. However, due to differences inglycosylation, the baculovirus system produces a protein, under reducingconditions, migrating at around 60 kD, while the mammalian systemgenerates a protein migrating at around 55 kD.

Additionally, fusion proteins of D-SLAM polypeptides, including thepredicted extracellular domain or the mature form of D-SLAM, can befused to FLAG for mammalian cell expression. For example, theextracellular domain can be amplifed using the following primers: a 5′primer containing a BamHI restriction site shown in bold:GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ ID NO: 22) anda 3′ primer, containing a Xba restriction site shown in bold:GCAGCATCTAGATTACTTGTCATCGTCGTCCTTGTAGTCATCTTTGTAGGAGGCCTTCCCTGGTGCTG(SEQ ID NO: 26).

Example 11 Production of an Antibody

a) Hybridoma Technology

The antibodies of the present invention can be prepared by a variety ofmethods. (See, Current Protocols, Chapter 2.) As one example of suchmethods, cells expressing D-SLAM is administered to an animal to inducethe production of sera containing polyclonal antibodies. In a preferredmethod, a preparation of D-SLAM protein is prepared and purified torender it substantially free of natural contaminants. Such a preparationis then introduced into an animal in order to produce polyclonalantisera of greater specific activity.

In the most preferred method, the antibodies of the present inventionare monoclonal antibodies (or protein binding fragments thereof). Suchmonoclonal antibodies can be prepared using hybridoma technology.(Köhler et al., Nature 256:495 (1975); Köhler et al., Eur. J. Immunol.6:511 (1976); Köhler et al., Eur. J. Immunol. 6:292 (1976); Hammerlinget al., in: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y.,pp. 563-681 (1981).) In general, such procedures involve immunizing ananimal (preferably a mouse) with D-SLAM polypeptide or, more preferably,with a secreted D-SLAM polypeptide-expressing cell. Such cells may becultured in any suitable tissue culture medium; however, it ispreferable to culture cells in Earle's modified Eagle's mediumsupplemented with 10% fetal bovine serum (inactivated at about 56° C.),and supplemented with about 10 g/l of nonessential amino acids, about1,000 U/ml of penicillin, and about 100 ug/ml of streptomycin.

The splenocytes of such mice are extracted and fused with a suitablemyeloma cell line. Any suitable myeloma cell line may be employed inaccordance with the present invention; however, it is preferable toemploy the parent myeloma cell line (SP20), available from the ATCC.After fusion, the resulting hybridoma cells are selectively maintainedin HAT medium, and then cloned by limiting dilution as described byWands et al. (Gastroenterology 80:225-232 (1981).) The hybridoma cellsobtained through such a selection are then assayed to identify cloneswhich secrete antibodies capable of binding the D-SLAM polypeptide.

Alternatively, additional antibodies capable of binding to D-SLAMpolypeptide can be produced in a two-step procedure using anti-idiotypicantibodies. Such a method makes use of the fact that antibodies arethemselves antigens, and therefore, it is possible to obtain an antibodywhich binds to a second antibody. In accordance with this method,protein specific antibodies are used to immunize an animal, preferably amouse. The splenocytes of such an animal are then used to producehybridoma cells, and the hybridoma cells are screened to identify cloneswhich produce an antibody whose ability to bind to the D-SLAMprotein-specific antibody can be blocked byD-SLAM. Such antibodiescomprise anti-idiotypic antibodies to the D-SLAM protein-specificantibody and can be used to immunize an animal to induce formation offurther D-SLAM protein-specific antibodies.

It will be appreciated that Fab and F(ab′)2 and other fragments of theantibodies of the present invention may be used according to the methodsdisclosed herein. Such fragments are typically produced by proteolyticcleavage, using enzymes such as papain (to produce Fab fragments) orpepsin (to produce F(ab′)2 fragments). Alternatively, secreted D-SLAMprotein-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry.

For in vivo use of antibodies in humans, it may be preferable to use“humanized” chimeric monoclonal antibodies. Such antibodies can beproduced using genetic constructs derived from hybridoma cells producingthe monoclonal antibodies described above. Methods for producingchimeric antibodies are known in the art. (See, for review, Morrison,Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabillyet al., U.S. Pat. No. 4,816,567; Taniguchi et al., EP 171496; Morrisonet al., EP 173494; Neuberger et al., WO 8601533; Robinson et al., WO8702671; Boulianne et al., Nature 312:643 (1984); Neuberger et al.,Nature 314:268 (1985).)

b) Isolation of Antibody Fragments Directed Against D-SLAM from aLibrary of scFvs.

Naturally occuring V-genes isolated from human PBLs are constructed intoa large library of antibody fragments which contain reactivities againstD-SLAM to which the donor may or may not have been exposed (see e.g.,U.S. Pat. No. 5,885,793 incorporated herein in its entirety byreference).

Rescue of the Library. A library of scFvs is constructed from the RNA ofhuman PBLs as described in WO92/01047. To rescue phage displayingantibody fragments, approximately 109 E. coli harbouring the phagemidare used to inoculate 50 ml of 2×TY containing 1% glucose and 100 ug/mlof ampicillin (2×TY-AMP-GLU) and grown to an O.D. of 0.8 with shaking.Five ml of this culture is used to innoculate 50 ml of 2×TY-AMP-GLU,2×10⁸ TU of delta gene 3 helper (M13 delta gene III, see WO92/01047) areadded and the culture incubated at 37° C. for 45 minutes without shakingand then at 37° C. for 45 minutes with shaking. The culture iscentrifuged at 4000 r.p.m. for 10 min. and the pellet resuspended in 2liters of of 2×TY containing 100 ug/ml ampicillin and 50 ug/ml kanamycinand grown overnight. Phage are prepared as described in WO92/01047.

M13 delta gene III is prepared as follows: M13 delta gene III helperphage does not encode gene III protein, hence the phage(mid) displayingantibody fragments have a greater avidity of binding to antigen.Infectious M13 delta gene III particles are made by growing the helperphage in cells harbouring a pUC19 derivative supplying the wild typegene III protein during phage morphogenesis. The culture is incubatedfor 1 hour at 37° C. without shaking and then for a further hour at 37°C. with shaking. Cells are spun down (IEC-Centra 8, 4000 revs/min for 10min), resuspended in 300 ml 2×TY broth containing 100 ug ampicillin/mland 25 ug kanamycin/ml (2×TY-AMP-KAN) and grown overnight, shaking at37° C. Phage particles are purified and concentrated from the culturemedium by two PEG-precipitations (Sambrook et al., 1990), resuspended in2 ml PBS and passed through a 0.45 um filter (Minisart NML; Sartorius)to give a final concentration of approximately 10¹³ transducing units/ml(ampicillin-resistant clones).

Panning of the Library. Immunotubes (Nunc) are coated overnight in PBSwith 4 ml of either 100 ug/ml or 10 ug/ml of a polypeptide of thepresent invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at37° C. and then washed 3 times in PBS. Approximately 10¹³ TU of phage isapplied to the tube and incubated for 30 minutes at room temperaturetumbling on an over and under turntable and then left to stand foranother 1.5 hours. Tubes are washed 10 times with PBS 0.1% Tween-20 and10 times with PBS. Phage are eluted by adding 1 ml of 100 mMtriethylamine and rotating 15 minutes on an under and over turntableafter which the solution is immediately neutralized with 0.5 ml of 1.0MTris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coliTG1 by incubating eluted phage with bacteria for 30 minutes at 37° C.The E. coli are then plated on TYE plates containing 1% glucose and 100ug/ml ampicillin. The resulting bacterial library is then rescued withdelta gene 3 helper phage as described above to prepare phage for asubsequent round of selection. This process is then repeated for a totalof 4 rounds of affinity purification with tube-washing increased to 20times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.

Characterization of Binders. Eluted phage from the 3rd and 4th rounds ofselection are used to infect E. coli HB 2151 and soluble scFv isproduced (Marks, et al., 1991) from single colonies for assay. ELISAsare performed with microtitre plates coated with either 10 pg/ml of thepolypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clonespositive in ELISA are further characterized by PCR fingerprinting (seee.g., WO92/01047) and then by sequencing.

Example 12 Production of D-SLAM Protein for High-Throughput ScreeningAssays

The following protocol produces a supernatant containing D-SLAMpolypeptide to be tested. This supernatant can then be used in theScreening Assays described in Examples 14-21.

First, dilute Poly-D-Lysine (644 587 Boehringer-Mannheim) stock solution(1 mg/ml in PBS) 1:20 in PBS (w/o calcium or magnesium 17-516FBiowhittaker) for a working solution of 50 ug/ml. Add 200 ul of thissolution to each well (24 well plates) and incubate at RT for 20minutes. Be sure to distribute the solution over each well (note: a12-channel pipetter may be used with tips on every other channel).Aspirate off the Poly-D-Lysine solution and rinse with 1 ml PBS(Phosphate Buffered Saline). The PBS should remain in the well untiljust prior to plating the cells and plates may be poly-lysine coated inadvance for up to two weeks.

Plate 293T cells (do not carry cells past P+20) at 2×10⁵ cells/well in0.5 ml DMEM (Dulbecco's Modified Eagle Medium)(with 4.5 G/L glucose andL-glutamine (12-604F Biowhittaker))/10% heat inactivated FBS (14-503FBiowhittaker)/1×Penstrep (17-602E Biowhittaker). Let the cells growovernight.

The next day, mix together in a sterile solution basin: 300 ulLipofectamine (18324-012 Gibco/BRL) and 5 ml Optimem I (31985070Gibco/BRL)/96-well plate. With a small volume multi-channel pipetter,aliquot approximately 2 ug of an expression vector containing apolynucleotide insert, produced by the methods described in Examples8-10, into an appropriately labeled 96-well round bottom plate. With amulti-channel pipetter, add 50 ul of the Lipofectamine/Optimem I mixtureto each well. Pipette up and down gently to mix. Incubate at RT 15-45minutes. After about 20 minutes, use a multi-channel pipetter to add 150ul Optimem I to each well. As a control, one plate of vector DNA lackingan insert should be transfected with each set of transfections.

Preferably, the transfection should be performed by tag-teaming thefollowing tasks. By tag-teaming, hands on time is cut in half, and thecells do not spend too much time on PBS. First, person A aspirates offthe media from four 24-well plates of cells, and then person B rinseseach well with 0.5-1 ml PBS. Person A then aspirates off PBS rinse, andperson B, using a 12-channel pipetter with tips on every other channel,adds the 200 ul of DNA/Lipofectamine/Optimem I complex to the odd wellsfirst, then to the even wells, to each row on the 24-well plates.Incubate at 37° C. for 6 hours.

While cells are incubating, prepare appropriate media, either 1% BSA inDMEM with 1×penstrep, or HGS CHO-5 media (116.6 mg/L of CaCl₂ (anhyd);0.00130 mg/L CuSO₄.5H₂O; 0.050 mg/L of Fe(NO₃)₃-9H₂O; 0.417 mg/L ofFeSO₄.7H₂O; 311.80 mg/L of Kcl; 28.64 mg/L of MgCl₂; 48.84 mg/L ofMgSO₄; 6995.50 mg/L of NaCl; 2400.0 mg/L of NaHCO₃; 62.50 mg/L ofNaH₂PO₄—H₂O; 71.02 mg/L of Na₂HPO₄; 0.4320 mg/L of ZnSO₄.7H₂O; 0.002mg/L of Arachidonic Acid; 1.022 mg/L of Cholesterol; 0.070 mg/L ofDL-alpha-Tocopherol-Acetate; 0.0520 mg/L of Linoleic Acid; 0.010 mg/L ofLinolenic Acid; 0.010 mg/L of Myristic Acid; 0.010 mg/L of Oleic Acid;0.010 mg/L of Palmitric Acid; 0.010 mg/L of Palmitic Acid; 100 mg/L ofPluronic F-68; 0.010 mg/L of Stearic Acid; 2.20 mg/L of Tween 80; 4551mg/L of D-Glucose; 130.85 mg/ml of L-Alanine; 147.50 mg/ml ofL-Arginine-HCL; 7.50 mg/ml of L-Asparagine-H₂O; 6.65 mg/ml of L-AsparticAcid; 29.56 mg/ml of L-Cystine-2HCL-H₂O; 31.29 mg/ml of L-Cystine-2HCL;7.35 mg/ml of L-Glutamic Acid; 365.0 mg/ml of L-Glutamine; 18.75 mg/mlof Glycine; 52.48 mg/ml of L-Histidine-HCL-H₂O; 106.97 mg/ml ofL-Isoleucine; 111.45 mg/ml of L-Leucine; 163.75 mg/ml of L-Lysine HCL;32.34 mg/ml of L-Methionine; 68.48 mg/ml of L-Phenylalainine; 40.0 mg/mlof L-Proline; 26.25 mg/ml of L-Serine; 101.05 mg/ml of L-Threonine;19.22 mg/ml of L-Tryptophan; 91.79 mg/ml of L-Tryrosine-2Na-2H₂O; and99.65 mg/ml of L-Valine; 0.0035 mg/L of Biotin; 3.24 mg/L of D-CaPantothenate; 11.78 mg/L of Choline Chloride; 4.65 mg/L of Folic Acid;15.60 mg/L of i-Inositol; 3.02 mg/L of Niacinamide; 3.00 mg/L ofPyridoxal HCL; 0.031 mg/L of Pyridoxine HCL; 0.319 mg/L of Riboflavin;3.17 mg/L of Thiamine HCL; 0.365 mg/L of Thymidine; 0.680 mg/L ofVitamin B₁₂; 25 mM of HEPES Buffer; 2.39 mg/L of Na Hypoxanthine; 0.105mg/L of Lipoic Acid; 0.081 mg/L of Sodium Putrescine-2HCL; 55.0 mg/L ofSodium Pyruvate; 0.0067 mg/L of Sodium Selenite; 20 uM of Ethanolamine;0.122 mg/L of Ferric Citrate; 41.70 mg/L of Methyl-B-Cyclodextrincomplexed with Linoleic Acid; 33.33 mg/L of Methyl-B-Cyclodextrincomplexed with Oleic Acid; 10 mg/L of Methyl-B-Cyclodextrin complexedwith Retinal Acetate. Adjust osmolarity to 327 mOsm) with 2 mm glutamineand 1×penstrep. (BSA (81-068-3 Bayer) 100 gm dissolved in 1 L DMEM for a10% BSA stock solution). Filter the media and collect 50 ul forendotoxin assay in 15 ml polystyrene conical.

The transfection reaction is terminated, preferably by tag-teaming, atthe end of the incubation period. Person A aspirates off thetransfection media, while person B adds 1.5 ml appropriate media to eachwell. Incubate at 37° C. for 45 or 72 hours depending on the media used:1% BSA for 45 hours or CHO-5 for 72 hours.

On day four, using a 300 ul multichannel pipetter, aliquot 600 ul in one1 ml deep well plate and the remaining supernatant into a 2 ml deepwell. The supernatants from each well can then be used in the assaysdescribed in Examples 14-21.

It is specifically understood that when activity is obtained in any ofthe assays described below using a supernatant, the activity originatesfrom either the D-SLAM polypeptide directly (e.g., as a secretedprotein) or by D-SLAM inducing expression of other proteins, which arethen secreted into the supernatant. Thus, the invention further providesa method of identifying the protein in the supernatant characterized byan activity in a particular assay.

Example 13 Construction of GAS Reporter Construct

One signal transduction pathway involved in the differentiation andproliferation of cells is called the Jaks-STATs pathway. Activatedproteins in the Jaks-STATs pathway bind to gamma activation site “GAS”elements or interferon-sensitive responsive element (“ISRE”), located inthe promoter of many genes. The binding of a protein to these elementsalter the expression of the associated gene.

GAS and ISRE elements are recognized by a class of transcription factorscalled Signal Transducers and Activators of Transcription, or “STATs.”There are six members of the STATs family. Stat1 and Stat3 are presentin many cell types, as is Stat2 (as response to IFN-alpha iswidespread). Stat4 is more restricted and is not in many cell typesthough it has been found in T helper class I, cells after treatment withIL-12. Stat5 was originally called mammary growth factor, but has beenfound at higher concentrations in other cells including myeloid cells.It can be activated in tissue culture cells by many cytokines.

The STATs are activated to translocate from the cytoplasm to the nucleusupon tyrosine phosphorylation by a set of kinases known as the JanusKinase (“Jaks”) family. Jaks represent a distinct family of solubletyrosine kinases and include Tyk2, Jak1, Jak2, and Jak3. These kinasesdisplay significant sequence similarity and are generally catalyticallyinactive in resting cells.

The Jaks are activated by a wide range of receptors summarized in theTable below. (Adapted from review by Schidler and Darnell, Ann. Rev.Biochem. 64:621-51 (1995).) A cytokine receptor family, capable ofactivating Jaks, is divided into two groups: (a) Class 1 includesreceptors for IL-2, IL-3, IL-4, IL-6, IL-7, IL-9, IL-11, IL-12, IL-15,Epo, PRL, GH, G-CSF, GM-CSF, LIF, CNTF, and thrombopoietin; and (b)Class 2 includes IFN-a, IFN-g, and IL-10. The Class 1 receptors share aconserved cysteine motif (a set of four conserved cysteines and onetryptophan) and a WSXWS motif (a membrane proxial region encodingTrp-Ser-D-SLAM-Trp-Ser (SEQ ID NO:5)).

Thus, on binding of a ligand to a receptor, Jaks are activated, which inturn activate STATs, which then translocate and bind to GAS elements.This entire process is encompassed in the Jaks-STATs signal transductionpathway.

Therefore, activation of the Jaks-STATs pathway, reflected by thebinding of the GAS or the ISRE element, can be used to indicate proteinsinvolved in the proliferation and differentiation of cells. For example,growth factors and cytokines are known to activate the Jaks-STATspathway. (See Table below.) Thus, by using GAS elements linked toreporter molecules, activators of the Jaks-STATs pathway can beidentified. JAKs Ligand tyk2 Jak1 Jak2 Jak3 STATS GAS(elements) or ISREIFN family IFN-a/B + + − − 1, 2, 3 ISRE IFN-g + + − 1 GAS (IRF1 > Lys6 >IFP) Il-10 + ? ? − 1, 3 gp130 family IL-6 (Pleiotrohic) + + + ? 1, 3 GAS(IRF1 > Lys6 > IFP) Il-11(Pleiotrohic) ? + ? ? 1, 3 OnM(Pleiotrohic)? + + ? 1, 3 LIF(Pleiotrohic) ? + + ? 1, 3 CNTF(Pleiotrohic) −/+ + + ?1, 3 G-CSF(Pleiotrohic) ? + ? ? 1, 3 IL-12(Pleiotrohic) + − + + 1, 3 g-Cfamily IL-2 (lymphocytes) − + − + 1, 3, 5 GAS IL-4 (lymph/myeloid) − +− + 6 GAS (IRF1 = IFP >> Ly6)(IgH) IL-7 (lymphocytes) − + − + 5 GAS IL-9(lymphocytes) − + − + 5 GAS IL-13 (lymphocyte ) − + ? ? 6 GAS IL-15 ? +? + 5 GAS gp140 family IL-3 (myeloid) − − + − 5 GAS (IRF1 > IFP >> Ly6)IL-5 (myeloid) − − + − 5 GAS GM-CSF (myeloid) − − + − 5 GAS Growthhormone family GH ? − + − 5 PRL ? +/− + − 1, 3, 5 EPO ? − + − 5GAS(B-CAS > IRF1 = IFP >> Ly6) Receptor Tyrosine Kinases EGF ? + + − 1,3 GAS (IRF1) PDGF ? + + − 1, 3 CSF-1 ? + + − 1, 3 GAS (not IRF1)

To construct a synthetic GAS containing promoter element, which is usedin the Biological Assays described in Examples 14-15, a PCR basedstrategy is employed to generate a GAS-SV40 promoter sequence. The 5′primer contains four tandem copies of the GAS binding site found in theIRF1 promoter and previously demonstrated to bind STATs upon inductionwith a range of cytokines (Rothman et al., Immunity 1:457-468 (1994).),although other GAS or ISRE elements can be used instead. The 5′ primeralso contains 18 bp of sequence complementary to the SV40 early promotersequence and is flanked with an XhoI site. The sequence of the 5′ primeris: 5′:GCGCCTCGAGATTTCCCCGAAATCTAGATTTCCC (SEQ ID NO:6)CGAAATGATTTCCCCGAAATGATTTCCCCGAAATATC TGCCATCTCAATTAG:3′.

The downstream primer is complementary to the SV40 promoter and isflanked with a Hind III site: 5′:GCGGCAAGCTTTTTGCAAAGCCTAGGC:3′ (SEQ IDNO:7).

PCR amplification is performed using the SV40 promoter template presentin the B-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI/Hind III and subcloned into BLSK2-.(Stratagene.) Sequencing with forward and reverse primers confirms thatthe insert contains the following sequence:5′:CTCGAGATTTCCCCGAAATCTAGATTTCCCCGAA (SEQ ID NO:8)ATGATTTCCCCGAAATGATTTCCCCGAAATATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGC TTTTGCAAAAAGCTT:3′.

With this GAS promoter element linked to the SV40 promoter, a GAS:SEAP2reporter construct is next engineered. Here, the reporter molecule is asecreted alkaline phosphatase, or “SEAP.” Clearly, however, any reportermolecule can be instead of SEAP, in this or in any of the otherExamples. Well known reporter molecules that can be used instead of SEAPinclude chloramphenicol acetyltransferase (CAT), luciferase, alkalinephosphatase, B-galactosidase, green fluorescent protein (GFP), or anyprotein detectable by an antibody.

The above sequence confirmed synthetic GAS-SV40 promoter element issubcloned into the pSEAP-Promoter vector obtained from Clontech usingHindIII and XhoI, effectively replacing the SV40 promoter with theamplified GAS:SV40 promoter element, to create the GAS-SEAP vector.However, this vector does not contain a neomycin resistance gene, andtherefore, is not preferred for mammalian expression systems.

Thus, in order to generate mammalian stable cell lines expressing theGAS-SEAP reporter, the GAS-SEAP cassette is removed from the GAS-SEAPvector using SalI and NotI, and inserted into a backbone vectorcontaining the neomycin resistance gene, such as pGFP-1 (Clontech),using these restriction sites in the multiple cloning site, to createthe GAS-SEAP/Neo vector. Once this vector is transfected into mammaliancells, this vector can then be used as a reporter molecule for GASbinding as described in Examples 14-15.

Other constructs can be made using the above description and replacingGAS with a different promoter sequence. For example, construction ofreporter molecules containing NFK-B and EGR promoter sequences aredescribed in Examples 16 and 17. However, many other promoters can besubstituted using the protocols described in these Examples. Forinstance, SRE, IL-2, NFAT, or Osteocalcin promoters can be substituted,alone or in combination (e.g., GAS/NF-KB/EGR, GAS/NF-KB, Il-2/NFAT, orNF-KB/GAS). Similarly, other cell lines can be used to test reporterconstruct activity, such as HELA (epithelial), HUVEC (endothelial), Reh(B-cell), Saos-2 (osteoblast), HUVAC (aortic), or Cardiomyocyte.

Example 14 High-Throughput Screening Assay for T-cell Activity

The following protocol is used to assess T-cell activity of D-SLAM bydetermining whether D-SLAM supernatant proliferates and/ordifferentiates T-cells. T-cell activity is assessed using theGAS/SEAP/Neo construct produced in Example 13. Thus, factors thatincrease SEAP activity indicate the ability to activate the Jaks-STATSsignal transduction pathway. The T-cell used in this assay is JurkatT-cells (ATCC Accession No. TIB-152), although Molt-3 cells (ATCCAccession No. CRL-1552) and Molt-4 cells (ATCC Accession No. CRL-1582)cells can also be used.

Jurkat T-cells are lymphoblastic CD4⁺ Th1 helper cells. In order togenerate stable cell lines, approximately 2 million Jurkat cells aretransfected with the GAS-SEAP/neo vector using DMRIE-C (LifeTechnologies)(transfection procedure described below). The transfectedcells are seeded to a density of approximately 20,000 cells per well andtransfectants resistant to 1 mg/ml genticin selected. Resistant coloniesare expanded and then tested for their response to increasingconcentrations of interferon gamma. The dose response of a selectedclone is demonstrated.

Specifically, the following protocol will yield sufficient cells for 75wells containing 200 ul of cells. Thus, it is either scaled up, orperformed in multiple to generate sufficient cells for multiple 96 wellplates. Jurkat cells are maintained in RPMI+10% serum with 1% Pen-Strep.Combine 2.5 mls of OPTI-MEM (Life Technologies) with 10 ug of plasmidDNA in a T25 flask. Add 2.5 ml OPTI-MEM containing 50 ul of DMRIE-C andincubate at room temperature for 15-45 mins.

During the incubation period, count cell concentration, spin down therequired number of cells (107 per transfection), and resuspend inOPTI-MEM to a final concentration of 10⁷ cells/ml. Then add 1 ml of1×10⁷ cells in OPTI-MEM to T25 flask and incubate at 37° C. for 6 hrs.After the incubation, add 10 ml of RPMI+15% serum.

The Jurkat:GAS-SEAP stable reporter lines are maintained in RPMI+10%serum, 1 mg/ml Genticin, and 1% Pen-Strep. These cells are treated withsupernatants containing D-SLAM polypeptides or D-SLAM inducedpolypeptides as produced by the protocol described in Example 12.

On the day of treatment with the supernatant, the cells should be washedand resuspended in fresh RPMI+10% serum to a density of 500,000 cellsper ml. The exact number of cells required will depend on the number ofsupernatants being screened. For one 96 well plate, approximately 10million cells (for 10 plates, 100 million cells) are required.

Transfer the cells to a triangular reservoir boat, in order to dispensethe cells into a 96 well dish, using a 12 channel pipette. Using a 12channel pipette, transfer 200 ul of cells into each well (thereforeadding 100,000 cells per well).

After all the plates have been seeded, 50 ul of the supernatants aretransferred directly from the 96 well plate containing the supernatantsinto each well using a 12 channel pipette. In addition, a dose ofexogenous interferon gamma (0.1, 1.0, 10 ng) is added to wells H9, H10,and H11 to serve as additional positive controls for the assay.

The 96 well dishes containing Jurkat cells treated with supernatants areplaced in an incubator for 48 hrs (note: this time is variable between48-72 hrs). 35 ul samples from each well are then transferred to anopaque 96 well plate using a 12 channel pipette. The opaque platesshould be covered (using sellophene covers) and stored at −20° C. untilSEAP assays are performed according to Example 18. The plates containingthe remaining treated cells are placed at 4° C. and serve as a source ofmaterial for repeating the assay on a specific well if desired.

As a positive control, 100 Unit/ml interferon gamma can be used which isknown to activate Jurkat T cells. Over 30 fold induction is typicallyobserved in the positive control wells.

Example 15 High-Throughput Screening Assay: Identifying Myeloid Activity

The following protocol is used to assess myeloid activity of D-SLAM bydetermining whether D-SLAM proliferates and/or differentiates myeloidcells. Myeloid cell activity is assessed using the GAS/SEAP/Neoconstruct produced in Example 13. Thus, factors that increase SEAPactivity indicate the ability to activate the Jaks-STATS signaltransduction pathway. The myeloid cell used in this assay is U937, apre-monocyte cell line, although TF-1, HL60, or KG1 can be used.

To transiently transfect U937 cells with the GAS/SEAP/Neo constructproduced in Example 13, a DEAE-Dextran method (Kharbanda et. al., 1994,Cell Growth & Differentiation, 5:259-265) is used. First, harvest 2×10e7U937 cells and wash with PBS. The U937 cells are usually grown in RPMI1640 medium containing 10% heat-inactivated fetal bovine serum (FBS)supplemented with 100 units/ml penicillin and 100 mg/ml streptomycin.

Next, suspend the cells in 1 ml of 20 mM Tris-HCl (pH 7.4) buffercontaining 0.5 mg/ml DEAE-Dextran, 8 ug GAS-SEAP2 plasmid DNA, 140 mMNaCl, 5 mM KCl, 375 uM Na₂HPO₄.7H₂₀, 1 mM MgCl₂, and 675 uM CaCl₂.Incubate at 37° C. for 45 min.

Wash the cells with RPMI 1640 medium containing 10% FBS and thenresuspend in 10 ml complete medium and incubate at 37° C. for 36 hr.

The GAS-SEAP/U937 stable cells are obtained by growing the cells in 400ug/ml G418. The G418-free medium is used for routine growth but everyone to two months, the cells should be re-grown in 400 ug/ml G418 forcouple of passages.

These cells are tested by harvesting 1×10⁸ cells (this is enough for ten96-well plates assay) and wash with PBS. Suspend the cells in 200 mlabove described growth medium, with a final density of 5×10⁵ cells/ml.Plate 200 ul cells per well in the 96-well plate (or 1×10⁵ cells/well).

Add 50 ul of the supernatant prepared by the protocol described inExample 12. Incubate at 37 degee C for 48 to 72 hr. As a positivecontrol, 100 Unit/ml interferon gamma can be used which is known toactivate U937 cells. Over 30 fold induction is typically observed in thepositive control wells. SEAP assay the supernatant according to theprotocol described in Example 18.

Example 16 High-Throughput Screening Assay: Identifying NeuronalActivity

When cells undergo differentiation and proliferation, a group of genesare activated through many different signal transduction pathways. Oneof these genes, EGR1 (early growth response gene 1), is induced invarious tissues and cell types upon activation. The promoter of EGR1 isresponsible for such induction. Using the EGR1 promoter linked toreporter molecules, activation of cells can be assessed by D-SLAM.

Particularly, the following protocol is used to assess neuronal activityin PC12 cell lines. PC12 cells (rat phenochromocytoma cells) are knownto proliferate and/or differentiate by activation with a number ofmitogens, such as TPA (tetradecanoyl phorbol acetate), NGF (nerve growthfactor), and EGF (epidermal growth factor). The EGR1 gene expression isactivated during this treatment. Thus, by stably transfecting PC12 cellswith a construct containing an EGR promoter linked to SEAP reporter,activation of PC12 cells by D-SLAM can be assessed.

The EGR/SEAP reporter construct can be assembled by the followingprotocol. The EGR-1 promoter sequence (−633 to +1)(Sakamoto K et al.,Oncogene 6:867-871 (1991)) can be PCR amplified from human genomic DNAusing the following primers: 5′ GCGCTCGAGGGATGACAGCGATAGAACCC (SEQ IDNO:9) CGG-3′ and 5′ GCGAAGCTTCGCGACTCCCCGGATCCGC (SEQ ID NO:10) CTC-3′.

Using the GAS:SEAP/Neo vector produced in Example 13, EGR1 amplifiedproduct can then be inserted into this vector. Linearize theGAS:SEAP/Neo vector using restriction enzymes XhoI/HindIII, removing theGAS/SV40 stuffer. Restrict the EGR1 amplified product with these sameenzymes. Ligate the vector and the EGR1 promoter.

To prepare 96 well-plates for cell culture, two mls of a coatingsolution (1:30 dilution of collagen type I (Upstate Biotech Inc.Cat#08-115) in 30% ethanol (filter sterilized)) is added per one 10 cmplate or 50 ml per well of the 96-well plate, and allowed to air dry for2 hr.

PC12 cells are routinely grown in RPMI-1640 medium (Bio Whittaker)containing 10% horse serum (JRH BIOSCIENCES, Cat. # 12449-78P), 5%heat-inactivated fetal bovine serum (FBS) supplemented with 100 units/mlpenicillin and 100 ug/ml streptomycin on a precoated 10 cm tissueculture dish. One to four split is done every three to four days. Cellsare removed from the plates by scraping and resuspended with pipettingup and down for more than 15 times.

Transfect the EGR/SEAP/Neo construct into PC12 using the Lipofectamineprotocol described in Example 12. EGR-SEAP/PC12 stable cells areobtained by growing the cells in 300 ug/ml G418. The G418-free medium isused for routine growth but every one to two months, the cells should bere-grown in 300 ug/ml G418 for couple of passages.

To assay for neuronal activity, a 10 cm plate with cells around 70 to80% confluent is screened by removing the old medium. Wash the cellsonce with PBS (Phosphate buffered saline). Then starve the cells in lowserum medium (RPMI-1640 containing 1% horse serum and 0.5% FBS withantibiotics) overnight.

The next morning, remove the medium and wash the cells with PBS. Scrapeoff the cells from the plate, suspend the cells well in 2 ml low serummedium. Count the cell number and add more low serum medium to reachfinal cell density as 5×10⁵ cells/ml.

Add 200 ul of the cell suspension to each well of 96-well plate(equivalent to 1×105 cells/well). Add 50 ul supernatant produced byExample 12, 37° C. for 48 to 72 hr. As a positive control, a growthfactor known to activate PC12 cells through EGR can be used, such as 50ng/ul of Neuronal Growth Factor (NGF). Over fifty-fold induction of SEAPis typically seen in the positive control wells. SEAP assay thesupernatant according to Example 18.

Example 17 High-Throughput Screening Assay for T-cell Activity

NF-KB (Nuclear Factor-kappaB) is a transcription factor activated by awide variety of agents including the inflammatory cytokines IL-1 andTNF, CD30 and CD40, lymphotoxin-alpha and lymphotoxin-beta, by exposureto LPS or thrombin, and by expression of certain viral gene products. Asa transcription factor, NF-kappaB regulates the expression of genesinvolved in immune cell activation, control of apoptosis (NF-kappaBappears to shield cells from apoptosis), B and T-cell development,anti-viral and antimicrobial responses, and multiple stress responses.

In non-stimulated conditions, NF-kappaB is retained in the cytoplasmwith I-kappaB (Inhibitor kappaB). However, upon stimulation, 1-kappaB isphosphorylated and degraded, causing NF-kappaB to shuttle to thenucleus, thereby activating transcription of target genes. Target genesactivated by NF-kappaB include IL-2, IL-6, GM-CSF, ICAM-1 and class 1MHC.

Due to its central role and ability to respond to a range of stimuli,reporter constructs utilizing the NF-kappaB promoter element are used toscreen the supernatants produced in Example 12. Activators or inhibitorsof NF-kappaB would be useful in treating, diagnosing, detecting, and/orpreventing diseases. For example, inhibitors of NF-kappaB could be usedto treat, diagnose, detect, and/or prevent those diseases related to theacute or chronic activation of NF-kappaB, such as rheumatoid arthritis.

To construct a vector containing the NF-kappaB promoter element, a PCRbased strategy is employed. The upstream primer contains four tandemcopies of the NF-kappaB binding site (GGGGACTTTCCC) (SEQ ID NO:11), 18bp of sequence complementary to the 5′ end of the SV40 early promotersequence, and is flanked with an XhoI site:5′GCGGCCTCGAGGGGACTTTCCCGGGGACTTTCCG (SEQ ID NO:12)GGGACTTTCCGGGACTTTCCATCCTGCCATCTCAA TTAG3′.

The downstream primer is complementary to the 3′ end of the SV40promoter and is flanked with a Hind III site: 5′-GCG GCA AGC TTT TTG CAAAGC CTA GGC-3′ (SEQ ID NO:7).

PCR amplification is performed using the SV40 promoter template presentin the pB-gal:promoter plasmid obtained from Clontech. The resulting PCRfragment is digested with XhoI and Hind III and subcloned into BLSK2-.(Stratagene) Sequencing with the T7 and T3 primers confirms the insertcontains the following sequence: 5′CTCGAGGGGACTTTCCCGGGGACTTTCCGGGGAC(SEQ ID NO:13) TTTCCGGGACTTTCCATCTGCCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAA AAGCTT3′.

Next, replace the SV40 minimal promoter element present in thepSEAP2-promoter plasmid (Clontech) with this NF-kappaB/SV40 fragmentusing XhoI and HindIII. However, this vector does not contain a neomycinresistance gene, and therefore, is not preferred for mammalianexpression systems.

In order to generate stable mammalian cell lines, theNF-kappaB/SV40/SEAP cassette is removed from the above NF-kappaB/SEAPvector using restriction enzymes SalI and NotI, and inserted into avector containing neomycin resistance. Particularly, theNF-kappaB/SV40/SEAP cassette was inserted into pGFP-1 (Clontech),replacing the GFP gene, after restricting pGFP-1 with SalI and NotI.

Once NF-kappaB/SV40/SEAP/Neo vector is created, stable Jurkat T-cellsare created and maintained according to the protocol described inExample 14. Similarly, the method for assaying supernatants with thesestable Jurkat T-cells is also described in Example 14. As a positivecontrol, exogenous TNF alpha (0.1, 1, 10 ng) is added to wells H9, H10,and H11, with a 5-10 fold activation typically observed.

Example 18 Assay for SEAP Activity

As a reporter molecule for the assays described in Examples 14-17, SEAPactivity is assayed using the Tropix Phospho-light Kit (Cat. BP-400)according to the following general procedure. The Tropix Phospho-lightKit supplies the Dilution, Assay, and Reaction Buffers used below.

Prime a dispenser with the 2.5× Dilution Buffer and dispense 15 ul of2.5× dilution buffer into Optiplates containing 35 ul of a supernatant.Seal the plates with a plastic sealer and incubate at 65° C. for 30 min.Separate the Optiplates to avoid uneven heating.

Cool the samples to room temperature for 15 minutes. Empty the dispenserand prime with the Assay Buffer. Add 50 microliters Assay Buffer andincubate at room temperature 5 min. Empty the dispenser and prime withthe Reaction Buffer (see the table below). Add 50 ul Reaction Buffer andincubate at room temperature for 20 minutes. Since the intensity of thechemiluminescent signal is time dependent, and it takes about 10 minutesto read 5 plates on luminometer, one should treat 5 plates at each timeand start the second set 10 minutes later.

Read the relative light unit in the luminometer. Set H12 as blank, andprint the results. An increase in chemiluminescence indicates reporteractivity. Reaction Buffer Formulation: # of plates Rxn buffer diluent(ml) CSPD (ml) 10 60 3 11 65 3.25 12 70 3.5 13 75 3.75 14 80 4 15 854.25 16 90 4.5 17 95 4.75 18 100 5 19 105 5.25 20 110 5.5 21 115 5.75 22120 6 23 125 6.25 24 130 6.5 25 135 6.75 26 140 7 27 145 7.25 28 150 7.529 155 7.75 30 160 8 31 165 8.25 32 170 8.5 33 175 8.75 34 180 9 35 1859.25 36 190 9.5 37 195 9.75 38 200 10 39 205 10.25 40 210 10.5 41 21510.75 42 220 11 43 225 11.25 44 230 11.5 45 235 11.75 46 240 12 47 24512.25 48 250 12.5 49 255 12.75 50 260 13

Example 19 High-Throughput Screening Assay Identifying Changes in SmallMolecule Concentration and Membrane Permeability

Binding of a ligand to a receptor is known to alter intracellular levelsof small molecules, such as calcium, potassium, sodium, and pH, as wellas alter membrane potential. These alterations can be measured in anassay to identify supernatants which bind to receptors of a particularcell. Although the following protocol describes an assay for calcium,this protocol can easily be modified to detect changes in potassium,sodium, pH, membrane potential, or any other small molecule which isdetectable by a fluorescent probe.

The following assay uses Fluorometric Imaging Plate Reader (“FLIPR”) tomeasure changes in fluorescent molecules (Molecular Probes) that bindsmall molecules. Clearly, any fluorescent molecule detecting a smallmolecule can be used instead of the calcium fluorescent molecule,fluo-3, used here.

For adherent cells, seed the cells at 10,000-20,000 cells/well in aCo-star black 96-well plate with clear bottom. The plate is incubated ina CO₂ incubator for 20 hours. The adherent cells are washed two times inBiotek washer with 200 ul of HBSS (Hank's Balanced Salt Solution)leaving 100 ul of buffer after the final wash.

A stock solution of 1 mg/ml fluo-3 is made in 10% pluronic acid DMSO. Toload the cells with fluo-3, 50 ul of 12 ug/ml fluo-3 is added to eachwell. The plate is incubated at 37° C. in a CO₂ incubator for 60 min.The plate is washed four times in the Biotek washer with HBSS leaving100 ul of buffer.

For non-adherent cells, the cells are spun down from culture media.Cells are re-suspended to 2-5×10⁶ cells/ml with HBSS in a 50-ml conicaltube. 4 ul of 1 mg/ml fluo-3 solution in 10% pluronic acid DMSO is addedto each ml of cell suspension. The tube is then placed in a 37° C. waterbath for 30-60 min. The cells are washed twice with HBSS, resuspended to1×10⁶ cells/ml, and dispensed into a microplate, 100 ul/well. The plateis centrifuged at 1000 rpm for 5 min. The plate is then washed once inDenley CellWash with 200 ul, followed by an aspiration step to 100 ulfinal volume.

For a non-cell based assay, each well contains a fluorescent molecule,such as fluo-3. The supernatant is added to the well, and a change influorescence is detected.

To measure the fluorescence of intracellular calcium, the FLIPR is setfor the following parameters: (1) System gain is 300-800 mW; (2)Exposure time is 0.4 second; (3) Camera F/stop is F/2; (4) Excitation is488 nm; (5) Emission is 530 nm; and (6) Sample addition is 50 ul.Increased emission at 530 nm indicates an extracellular signaling eventcaused by the a molecule, either D-SLAM or a molecule induced by D-SLAM,which has resulted in an increase in the intracellularCa++concentration.

Example 20 High-Throughput Screening Assay: Identifying Tyrosine KinaseActivity

The Protein Tyrosine Kinases (PTK) represent a diverse group oftransmembrane and cytoplasmic kinases. Within the Receptor ProteinTyrosine Kinase RPTK) group are receptors for a range of mitogenic andmetabolic growth factors including the PDGF, FGF, EGF, NGF, HGF andInsulin receptor subfamilies. In addition there are a large family ofRPTKs for which the corresponding ligand is unknown. Ligands for RPTKsinclude mainly secreted small proteins, but also membrane-bound andextracellular matrix proteins.

Activation of RPTK by ligands involves ligand-mediated receptordimerization, resulting in transphosphorylation of the receptor subunitsand activation of the cytoplasmic tyrosine kinases. The cytoplasmictyrosine kinases include receptor associated tyrosine kinases of thesrc-family (e.g., src, yes, Ick, lyn, fyn) and non-receptor linked andcytosolic protein tyrosine kinases, such as the Jak family, members ofwhich mediate signal transduction triggered by the cytokine superfamilyof receptors (e.g., the Interleukins, Interferons, GM-CSF, and Leptin).

Because of the wide range of known factors capable of stimulatingtyrosine kinase activity, identifying whether D-SLAM or a moleculeinduced by D-SLAM is capable of activating tyrosine kinase signaltransduction pathways is of interest. Therefore, the following protocolis designed to identify such molecules capable of activating thetyrosine kinase signal transduction pathways.

Seed target cells (e.g., primary keratinocytes) at a density ofapproximately 25,000 cells per well in a 96 well Loprodyne Silent ScreenPlates purchased from Nalge Nunc (Naperville, Ill.). The plates aresterilized with two 30 minute rinses with 100% ethanol, rinsed withwater and dried overnight. Some plates are coated for 2 hr with 100 mlof cell culture grade type I collagen (50 mg/ml), gelatin (2%) orpolylysine (50 mg/ml), all of which can be purchased from SigmaChemicals (St. Louis, Mo.) or 10% Matrigel purchased from BectonDickinson (Bedford, Mass.), or calf serum, rinsed with PBS and stored at4° C. Cell growth on these plates is assayed by seeding 5,000 cells/wellin growth medium and indirect quantitation of cell number through use ofalamarBlue as described by the manufacturer Alamar Biosciences, Inc.(Sacramento, Calif.) after 48 hr. Falcon plate covers #3071 from BectonDickinson (Bedford, Mass.) are used to cover the Loprodyne Silent ScreenPlates. Falcon Microtest III cell culture plates can also be used insome proliferation experiments.

To prepare extracts, A431 cells are seeded onto the nylon membranes ofLoprodyne plates (20,000/200 ml/well) and cultured overnight in completemedium. Cells are quiesced by incubation in serum-free basal medium for24 hr. After 5-20 minutes treatment with EGF (60 ng/ml) or 50 ul of thesupernatant produced in Example 12, the medium was removed and 100 ml ofextraction buffer ((20 mM HEPES pH 7.5, 0.15 M NaCl, 1% Triton X-100,0.1% SDS, 2 mM Na3VO4, 2 mM Na4P207 and a cocktail of proteaseinhibitors (# 1836170) obtained from Boeheringer Mannheim (Indianapolis,Ind.) is added to each well and the plate is shaken on a rotating shakerfor 5 minutes at 4° C. The plate is then placed in a vacuum transfermanifold and the extract filtered through the 0.45 mm membrane bottomsof each well using house vacuum. Extracts are collected in a 96-wellcatch/assay plate in the bottom of the vacuum manifold and immediatelyplaced on ice. To obtain extracts clarified by centrifugation, thecontent of each well, after detergent solubilization for 5 minutes, isremoved and centrifuged for 15 minutes at 4° C. at 16,000×g.

Test the filtered extracts for levels of tyrosine kinase activity.Although many methods of detecting tyrosine kinase activity are known,one method is described here.

Generally, the tyrosine kinase activity of a supernatant is evaluated bydetermining its ability to phosphorylate a tyrosine residue on aspecific substrate (a biotinylated peptide). Biotinylated peptides thatcan be used for this purpose include PSK1 (corresponding to amino acids6-20 of the cell division kinase cdc2-p34) and PSK2 (corresponding toamino acids 1-17 of gastrin). Both peptides are substrates for a rangeof tyrosine kinases and are available from Boehringer Mannheim.

The tyrosine kinase reaction is set up by adding the followingcomponents in order. First, add 10 ul of 5 uM Biotinylated Peptide, then10 ul ATP/Mg₂₊ (5 mM ATP/50 mM MgCl₂), then 10 ul of 5× Assay Buffer (40mM imidazole hydrochloride, pH7.3, 40 mM beta-glycerophosphate, 1 mMEGTA, 100 mM MgCl₂, 5 mM MnCl₂, 0.5 mg/ml BSA), then 5 ul of SodiumVanadate (1 mM), and then 5 ul of water. Mix the components gently andpreincubate the reaction mix at 30° C. for 2 min. Initial the reactionby adding 10 ul of the control enzyme or the filtered supernatant.

The tyrosine kinase assay reaction is then terminated by adding 10 ul of120 mm EDTA and place the reactions on ice.

Tyrosine kinase activity is determined by transferring 50 ul aliquot ofreaction mixture to a microtiter plate (MTP) module and incubating at37° C. for 20 min. This allows the streptavadin coated 96 well plate toassociate with the biotinylated peptide. Wash the MTP module with 300ul/well of PBS four times. Next add 75 ul of anti-phospotyrosineantibody conjugated to horse radish peroxidase (anti-P-Tyr-POD (0.5u/ml)) to each well and incubate at 37° C. for one hour. Wash the wellas above.

Next add 100 ul of peroxidase substrate solution (Boehringer Mannheim)and incubate at room temperature for at least 5 mins (up to 30 min).Measure the absorbance of the sample at 405 nm by using ELISA reader.The level of bound peroxidase activity is quantitated using an ELISAreader and reflects the level of tyrosine kinase activity.

Example 21 High-Throughput Screening Assay Identifying PhosphorylationActivity

As a potential alternative and/or compliment to the assay of proteintyrosine kinase activity described in Example 20, an assay which detectsactivation (phosphorylation) of major intracellular signal transductionintermediates can also be used. For example, as described below oneparticular assay can detect tyrosine phosphorylation of the Erk-1 andErk-2 kinases. However, phosphorylation of other molecules, such as Raf,JNK, p38 MAP, Map kinase kinase (MEK), MEK kinase, Src, Muscle specifickinase (MuSK), IRAK, Tec, and Janus, as well as any other phosphoserine,phosphotyrosine, or phosphothreonine molecule, can be detected bysubstituting these molecules for Erk-1 or Erk-2 in the following assay.

Specifically, assay plates are made by coating the wells of a 96-wellELISA plate with 0.1 ml of protein G (1 ug/ml) for 2 hr at room temp,(RT). The plates are then rinsed with PBS and blocked with 3% BSA/PBSfor 1 hr at RT. The protein G plates are then treated with 2 commercialmonoclonal antibodies (10 ng/well) against Erk-1 and Erk-2 (1 hr at RT)(Santa Cruz Biotechnology). (To detect other molecules, this step caneasily be modified by substituting a monoclonal antibody detecting anyof the above described molecules.) After 3-5 rinses with PBS, the platesare stored at 4° C. until use.

A431 cells are seeded at 20,000/well in a 96-well Loprodyne filterplateand cultured overnight in growth medium. The cells are then starved for48 hr in basal medium (DMEM) and then treated with EGF (6 ng/well) or 50ul of the supernatants obtained in Example 12 for 5-20 minutes. Thecells are then solubilized and extracts filtered directly into the assayplate.

After incubation with the extract for 1 hr at RT, the wells are againrinsed. As a positive control, a commercial preparation of MAP kinase(10 ng/well) is used in place of A431 extract. Plates are then treatedwith a commercial polyclonal (rabbit) antibody (1 ug/ml) whichspecifically recognizes the phosphorylated epitope of the Erk-1 andErk-2 kinases (1 hr at RT). This antibody is biotinylated by standardprocedures. The bound polyclonal antibody is then quantitated bysuccessive incubations with Europium-streptavidin and Europiumfluorescence enhancing reagent in the Wallac DELFIA instrument(time-resolved fluorescence). An increased fluorescent signal overbackground indicates a phosphorylation by D-SLAM or a molecule inducedby D-SLAM.

Example 22 Method of Determining Alterations in the D-SLAM Gene

RNA isolated from entire families or individual patients presenting witha phenotype of interest (such as a disease) is be isolated. cDNA is thengenerated from these RNA samples using protocols known in the art. (See,Sambrook.) The cDNA is then used as a template for PCR, employingprimers surrounding regions of interest in SEQ ID NO:1. Suggested PCRconditions consist of 35 cycles at 95° C. for 30 seconds; 60-120 secondsat 52-58° C.; and 60-120 seconds at 70° C., using buffer solutionsdescribed in Sidransky, D., et al., Science 252:706 (1991).

PCR products are then sequenced using primers labeled at their 5′ endwith T4 polynucleotide kinase, employing SequiTherm Polymerase.(Epicentre Technologies). The intron-exon borders of selected exons ofD-SLAM is also determined and genomic PCR products analyzed to confirmthe results. PCR products harboring suspected mutations in D-SLAM isthen cloned and sequenced to validate the results of the directsequencing.

PCR products of D-SLAM are cloned into T-tailed vectors as described inHolton, T. A. and Graham, M. W., Nucleic Acids Research, 19:1156 (1991)and sequenced with T7 polymerase (United States Biochemical). Affectedindividuals are identified by mutations in D-SLAM not present inunaffected individuals.

Genomic rearrangements are also observed as a method of determiningalterations in the D-SLAM gene. Genomic clones isolated according toExample 2 are nick-translated with digoxigenindeoxy-uridine5′-triphosphate (Boehringer Manheim), and FISH performed as described inJohnson, Cg. et al., Methods Cell Biol. 35:73-99 (1991). Hybridizationwith the labeled probe is carried out using a vast excess of human cot-1DNA for specific hybridization to the D-SLAM genomic locus.

Chromosomes are counterstained with 4,6-diamino-2-phenylidole andpropidium iodide, producing a combination of C- and R-bands. Alignedimages for precise mapping are obtained using a triple-band filter set(Chroma Technology, Brattleboro, Vt.) in combination with a cooledcharge-coupled device camera (Photometrics, Tucson, Ariz.) and variableexcitation wavelength filters. (Johnson, Cv. et al., Genet. Anal. Tech.Appl., 8:75 (1991).) Image collection, analysis and chromosomalfractional length measurements are performed using the ISee GraphicalProgram System. (Inovision Corporation, Durham, N.C.) Chromosomealterations of the genomic region of D-SLAM (hybridized by the probe)are identified as insertions, deletions, and translocations. TheseD-SLAM alterations are used as a diagnostic marker for an associateddisease.

Example 23 Method of Detecting Abnormal Levels of D-SLAM in a BiologicalSample

D-SLAM polypeptides can be detected in a biological sample, and if anincreased or decreased level of D-SLAM is detected, this polypeptide isa marker for a particular phenotype. Methods of detection are numerous,and thus, it is understood that one skilled in the art can modify thefollowing assay to fit their particular needs.

For example, antibody-sandwich ELISAs are used to detect D-SLAM in asample, preferably a biological sample. Wells of a microtiter plate arecoated with specific antibodies to D-SLAM, at a final concentration of0.2 to 10 ug/ml. The antibodies are either monoclonal or polyclonal andare produced by the method described in Example 11. The wells areblocked so that non-specific binding of D-SLAM to the well is reduced.

The coated wells are then incubated for >2 hours at RT with a samplecontaining D-SLAM. Preferably, serial dilutions of the sample should beused to validate results. The plates are then washed three times withdeionized or distilled water to remove unbounded D-SLAM.

Next, 50 ul of specific antibody-alkaline phosphatase conjugate, at aconcentration of 25-400 ng, is added and incubated for 2 hours at roomtemperature. The plates are again washed three times with deionized ordistilled water to remove unbounded conjugate.

Add 75 ul of 4-methylumbelliferyl phosphate (MUP) or p-nitrophenylphosphate (NPP) substrate solution to each well and incubate 1 hour atroom temperature. Measure the reaction by a microtiter plate reader.Prepare a standard curve, using serial dilutions of a control sample,and plot D-SLAM polypeptide concentration on the X-axis (log scale) andfluorescence or absorbance of the Y-axis (linear scale). Interpolate theconcentration of the D-SLAM in the sample using the standard curve.

Example 24 Formulating a Polypeptide

The D-SLAM composition will be formulated and dosed in a fashionconsistent with good medical practice, taking into account the clinicalcondition of the individual patient (especially the side effects oftreatment with the D-SLAM polypeptide alone), the site of delivery, themethod of administration, the scheduling of administration, and otherfactors known to practitioners. The “effective amount” for purposesherein is thus determined by such considerations.

As a general proposition, the total pharmaceutically effective amount ofD-SLAM administered parenterally per dose will be in the range of about1 ug/kg/day to 10 mg/kg/day of patient body weight, although, as notedabove, this will be subject to therapeutic discretion. More preferably,this dose is at least 0.01 mg/kg/day, and most preferably for humansbetween about 0.01 and 1 mg/kg/day for the hormone. If givencontinuously, D-SLAM is typically administered at a dose rate of about 1ug/kg/hour to about 50 ug/kg/hour, either by 1-4 injections per day orby continuous subcutaneous infusions, for example, using a mini-pump. Anintravenous bag solution may also be employed. The length of treatmentneeded to observe changes and the interval following treatment forresponses to occur appears to vary depending on the desired effect.

Effective dosages of the compositions of the present invention to beadministered may be determined through procedures well known to those inthe art which address such parameters as biological half-life,bioavailability, and toxicity. Such determination is well within thecapability of those skilled in the art, especially in light of thedetailed disclosure provided herein.

Bioexposure of an organism to D-SLAM polypeptide during therapy may alsoplay an important role in determining a therapeutically and/orpharmacologically effective dosing regime. Variations of dosing such asrepeated administrations of a relatively low dose of D-SLAM polypeptidefor a relatively long period of time may have an effect which istherapeutically and/or pharmacologically distinguishable from thatachieved with repeated administrations of a relatively high dose ofD-SLAM for a relatively short period of time.

Using the equivalent surface area dosage conversion factors supplied byFreireich, E. J., et al. (Cancer Chemotherapy Reports 50(4):219-44(1966)), one of ordinary skill in the art is able to convenientlyconvert data obtained from the use of D-SLAM in a given experimentalsystem into an accurate estimation of a pharmaceutically effectiveamount of D-SLAM polypeptide to be administered per dose in anotherexperimental system. Experimental data obtained through theadministration of D-SLAM may converted through the conversion factorssupplied by Freireich, et al., to accurate estimates of pharmaceuticallyeffective doses of D-SLAM in rat, monkey, dog, and human. The followingconversion table (Table III) is a summary of the data provided byFreireich, et al. Table III gives approximate factors for convertingdoses expressed in terms of mg/kg from one species to an equivalentsurface area dose expressed as mg/kg in another species tabulated. TABLEIII Equivalent Surface Area Dosage Conversion Factors. TO Mouse RatMonkey Dog FROM (20 g) (150 g) (3.5 kg)(8 kg) (60 kg) Human Mouse 1 ½ ¼⅙ {fraction ( 1/12)} Rat 2 1 ½ ¼ {fraction (1/7)} Monkey 4 2 1 ⅗ ⅓ Dog 64 {fraction (5/3)} 1 ½ Human 12  7 3 2 1

Thus, for example, using the conversion factors provided in Table III, adose of 50 mg/kg in the mouse converts to an appropriate dose of 12.5mg/kg in the monkey because (50 mg/kg)×(¼)=12.5 mg/kg. As an additionalexample, doses of 0.02, 0.08, 0.8, 2, and 8 mg/kg in the mouse equate toeffect doses of 1.667 micrograms/kg, 6.67 micrograms/kg, 66.7micrograms/kg, 166.7 micrograms/kg, and 0.667 mg/kg, respectively, inthe human.

Pharmaceutical compositions containing D-SLAM are administered orally,rectally, parenterally, intracistemally, intravaginally,intraperitoneally, topically (as by powders, ointments, gels, drops ortransdermal patch), bucally, or as an oral or nasal spray. In oneembodiment, “pharmaceutically acceptable carrier” means a non-toxicsolid, semisolid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. In a specific embodiment,“pharmaceutically acceptable” means approved by a regulatory agency ofthe federal or a state government or listed in the U.S. Pharmacopeia orother generally recognized pharmacopeia for use in animals, and moreparticularly humans. Nonlimiting examples of suitable pharmaceuticalcarriers according to this embodiment are provided in “Remington'sPharmaceutical Sciences” by E. W. Martin, and include sterile liquids,such as water and oils, including those of petroleum, animal, vegetableor synthetic origin, such as peanut oil, soybean oil, mineral oil,sesame oil and the like. Water is a preferred carrier when thepharmaceutical composition is administered intravenously. Salinesolutions and aqueous dextrose and glycerol solutions can be employed asliquid carriers, particularly for injectable solutions. The composition,if desired, can also contain minor amounts of wetting or emulsifyingagents, or pH buffering agents. These compositions can take the form ofsolutions, suspensions, emulsion, tablets, pills, capsules, powders,sustained-release formulations and the like.

The term “parenteral” as used herein refers to modes of administrationwhich include intravenous, intramuscular, intraperitoneal, intrasternal,subcutaneous and intraarticular injection and infusion.

In a preferred embodiment, D-SLAM compositions of the invention(including polypeptides, polynucleotides, and antibodies, and agonistsand/or antagonists thereof) are administered subcutaneously.

In another preferred embodiment, D-SLAM compositions of the invention(including polypeptides, polynucleotides, and antibodies, and agonistsand/or antagonists thereof) are administered intravenously.

D-SLAM compositions of the invention are also suitably administered bysustained-release systems. Suitable examples of sustained-releasecompositions include suitable polymeric materials (such as, for example,semi-permeable polymer matrices in the form of shaped articles, e.g.,films, or mirocapsules), suitable hydrophobic materials (for example asan emulsion in an acceptable oil) or ion exchange resins, and sparinglysoluble derivatives (such as, for example, a sparingly soluble salt).

Sustained-release matrices include polylactides (U.S. Pat. No.3,773,919, EP 58,481), copolymers of L-glutamic acid andgamma-ethyl-L-glutamate (Sidman, U. et al., Biopolymers 22:547-556(1983)), poly (2-hydroxyethyl methacrylate) (R. Langer et al., J.Biomed. Mater. Res. 15:167-277 (1981), and R. Langer, Chem. Tech.12:98-105 (1982)), ethylene vinyl acetate (R. Langer et al., Id.) orpoly-D-(−)-3-hydroxybutyric acid (EP 133,988).

In a preferred embodiment, D-SLAM compositions of the invention areformulated in a biodegradable, polymeric drug delivery system, forexample as described in U.S. Pat. Nos. 4,938,763; 5,278,201; 5,278,202;5,324,519; 5,340,849; and 5,487,897 and in International PublicationNumbers WO01/35929, WO00/24374, and WO0/06117 which are herebyincorporated by reference in their entirety. In specific preferredembodiments the D-SLAM compositions of the invention are formulatedusing the ATRIGEL® Biodegradable System of Atrix Laboratories, Inc.(Fort Collins, Colo.).

Examples of biodegradable polymers which can be used in the formulationof D-SLAM compositions, include but are not limited to, polylactides,polyglycolides, polycaprolactones, polyanhydrides, polyamides,polyurethanes, polyesteramides, polyorthoesters, polydioxanones,polyacetals, polyketals, polycarbonates, polyorthocarbonates,polyphosphazenes, polyhydroxybutyrates, polyhydroxyvalerates,polyalkylene oxalates, polyalkylene succinates, poly(malic acid),poly(amino acids), poly(methyl vinyl ether), poly(maleic anhydride),polyvinylpyrrolidone, polyethylene glycol, polyhydroxycellulose, chitin,chitosan, and copolymers, terpolymers, or combinations or mixtures ofthe above materials. The preferred polymers are those that have a lowerdegree of crystallization and are more hydrophobic. These polymers andcopolymers are more soluble in the biocompatible solvents than thehighly crystalline polymers such as polyglycolide and chitin which alsohave a high degree of hydrogen-bonding. Preferred materials with thedesired solubility parameters are the polylactides, polycaprolactones,and copolymers of these with glycolide in which there are more amorphousregions to enhance solubility. In specific preferred embodiments, thebiodegradable polymers which can be used in the formulation of D-SLAMcompositions are poly(lactide-co-glycolides). Polymer properties such asmolecular weight, hydrophobicity, and lactide/glycolide ratio may bemodified to obtain the desired drug D-SLAM release profile (See, e.g.,Ravivarapu et al., Journal of Pharmaceutical Sciences 89:732-741 (2000),which is hereby incorporated by reference in its entirety).

It is also preferred that the solvent for the biodegradable polymer benon-toxic, water miscible, and otherwise biocompatible. Examples of suchsolvents include, but are not limited to, N-methyl-2-pyrrolidone,2-pyrrolidone, C2 to C6 alkanols, C1 to C15 alchohols, dils, triols, andtetraols such as ethanol, glycerine propylene glycol, butanol; C3 to C15alkyl ketones such as acetone, diethyl ketone and methyl ethyl ketone;C3 to C15 esters such as methyl acetate, ethyl acetate, ethyl lactate;alkyl ketones such as methyl ethyl ketone, C1 to C15 amides such asdimethylformamide, dimethylacetamide and caprolactam; C3 to C20 etherssuch as tetrahydrofuran, or solketal; tweens, triacetin, propylenecarbonate, decylmethylsulfoxide, dimethyl sulfoxide, oleic acid,1-dodecylazacycloheptan-2-one, Other preferred solvents are benzylalchohol, benzyl benzoate, dipropylene glycol, tributyrin, ethyl oleate,glycerin, glycofural, isopropyl myristate, isopropyl palmitate, oleicacid, polyethylene glycol, propylene carbonate, and triethyl citrate.The most preferred solvents are

N-methyl-2-pyrrolidone, 2-pyrrolidone, dimethyl sulfoxide, triacetin,and propylene carbonate because of the solvating ability and theircompatibility.

Additionally, formulations comprising D-SLAM compositions and abiodegradable polymer may also include release-rate modification agentsand/or pore-forming agents. Examples of release-rate modification agentsinclude, but are not limited to, fatty acids, triglycerides, other likehydrophobic compounds, organic solvents, plasticizing compounds andhydrophilic compounds. Suitable release rate modification agentsinclude, for example, esters of mono-, di-, and tricarboxylic acids,such as 2-ethoxyethyl acetate, methyl acetate, ethyl acetate, diethylphthalate, dimethyl phthalate, dibutyl phthalate, dimethyl adipate,dimethyl succinate, dimethyl oxalate, dimethyl citrate, triethylcitrate, acetyl tributyl citrate, acetyl triethyl citrate, glyceroltriacetate, di(n-butyl) sebecate, and the like; polyhydroxy alcohols,such as propylene glycol, polyethylene glycol, glycerin, sorbitol, andthe like; fatty acids; triesters of glycerol, such as triglycerides,epoxidized soybean oil, and other epoxidized vegetable oils; sterols,such as cholesterol; alcohols, such as C.sub.6-C.sub.12 alkanols,2-ethoxyethanol. The release rate modification agent may be used singlyor in combination with other such agents. Suitable combinations ofrelease rate modification agents include, but are not limited to,glycerin/propylene glycol, sorbitol/glycerine, ethylene oxide/propyleneoxide, butylene glycol/adipic acid, and the like. Preferred release ratemodification agents include, but are not limited to, dimethyl citrate,triethyl citrate, ethyl heptanoate, glycerin, and hexanediol. Suitablepore-forming agents that may be used in the polymer composition include,but are not limited to, sugars such as sucrose and dextrose, salts suchas sodium chloride and sodium carbonate, polymers such ashydroxylpropylcellulose, carboxymethylcellulose, polyethylene glycol,and polyvinylpyrrolidone. Solid crystals that will provide a definedpore size, such as salt or sugar, are preferred.

In specific preferred embodiments the D-SLAM compositions of theinvention are formulated using the BEMA™ BioErodible MucoadhesiveSystem, MCA™ MucoCutaneous Absorption System, SMP™ Solvent MicroParticleSystem, or BCP™ BioCompatible Polymer System of Atrix Laboratories, Inc.(Fort Collins, Colo.).

Sustained-release compositions also include liposomally entrappedcompositions of the invention (see generally, Langer, Science249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy ofInfectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss,New York, pp. 317-327 and 353-365 (1989)). Liposomes containing D-SLAMpolypeptide my be prepared by methods known per se: DE 3,218,121;Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwanget al., Proc. Natl. Acad. Sci. (USA) 77:4030-4034 (1980); EP 52,322; EP36,676; EP 88,046; EP 143,949; EP 142,641; Japanese Pat. Appl.83-118008; U.S. Pat. Nos. 4,485,045 and 4,544,545; and EP 102,324.Ordinarily, the liposomes are of the small (about 200-800 Angstroms)unilamellar type in which the lipid content is greater than about 30mol. percent cholesterol, the selected proportion being adjusted for theoptimal D-SLAM polypeptide therapy.

In another embodiment systained release compositions of the inventioninclude crystal formulations known in the art.

In yet an additional embodiment, the compositions of the invention aredelivered by way of a pump (see Langer, supra; Sefton, CRC Crit. Ref.Biomed. Eng. 14:201 (1987); Buchwald et al., Surgery 88:507 (1980);Saudek et al., N. Engl. J. Med. 321:574 (1989)).

Other controlled release systems are discussed in the review by Langer(Science 249:1527-1533 (1990)).

For parenteral administration, in one embodiment, D-SLAM is formulatedgenerally by mixing it at the desired degree of purity, in a unit dosageinjectable form (solution, suspension, or emulsion), with apharmaceutically acceptable carrier, i.e., one that is non-toxic torecipients at the dosages and concentrations employed and is compatiblewith other ingredients of the formulation. For example, the formulationpreferably does not include oxidizing agents and other compounds thatare known to be deleterious to polypeptides.

Generally, the formulations are prepared by contacting D-SLAM uniformlyand intimately with liquid carriers or finely divided solid carriers orboth. Then, if necessary, the product is shaped into the desiredformulation. Preferably the carrier is a parenteral carrier, morepreferably a solution that is isotonic with the blood of the recipient.Examples of such carrier vehicles include water, saline, Ringer'ssolution, and dextrose solution. Non-aqueous vehicles such as fixed oilsand ethyl oleate are also useful herein, as well as liposomes.

The carrier suitably contains minor amounts of additives such assubstances that enhance isotonicity and chemical stability. Suchmaterials are non-toxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, succinate,acetic acid, and other organic acids or their salts; antioxidants suchas ascorbic acid; low molecular weight (less than about ten residues)polypeptides, e.g., polyarginine or tripeptides; proteins, such as serumalbumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids, such as glycine, glutamic acid,aspartic acid, or arginine; monosaccharides, disaccharides, and othercarbohydrates including cellulose or its derivatives, glucose, manose,or dextrins; chelating agents such as EDTA; sugar alcohols such asmannitol or sorbitol; counterions such as sodium; and/or nonionicsurfactants such as polysorbates, poloxamers, or PEG.

D-SLAM is typically formulated in such vehicles at a concentration ofabout 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3to 8. It will be understood that the use of certain of the foregoingexcipients, carriers, or stabilizers will result in the formation ofpolypeptide salts.

D-SLAM used for therapeutic administration can be sterile. Sterility isreadily accomplished by filtration through sterile filtration membranes(e.g., 0.2 micron membranes). Therapeutic polypeptide compositionsgenerally are placed into a container having a sterile access port, forexample, an intravenous solution bag or vial having a stopper pierceableby a hypodermic injection needle.

D-SLAM polypeptides ordinarily will be stored in unit or multi-dosecontainers, for example, sealed ampoules or vials, as an aqueoussolution or as a lyophilized formulation for reconstitution. As anexample of a lyophilized formulation, 10-ml vials are filled with 5 mlof sterile-filtered 1% (w/v) aqueous D-SLAM polypeptide solution, andthe resulting mixture is lyophilized. The infusion solution is preparedby reconstituting the lyophilized D-SLAM polypeptide usingbacteriostatic Water-for-Injection.

The invention also provides a pharmaceutical pack or kit comprising oneor more containers filled with one or more of the ingredients of thepharmaceutical compositions of the invention. Associated with suchcontainer(s) can be a notice in the form prescribed by a governmentalagency regulating the manufacture, use or sale of pharmaceuticals orbiological products, which notice reflects approval by the agency ofmanufacture, use or sale for human administration. In addition, D-SLAMmay be employed in conjunction with other therapeutic compounds.

The compositions of the invention may be administered alone or incombination with other therapeutic agents. Therapeutic agents that maybe administered in combination with the compositions of the invention,include but not limited to, members of the TNF family (e.g.,Neutrokine-Alpha and/or Neutrokine-AlphaSV (International ApplicationNo. PCT/US96/17957) or antagonists thereof), chemotherapeutic agents,antibiotics, steroidal and non-steroidal anti-inflammatories,conventional immunotherapeutic agents, cytokines and/or growth factors.Combinations may be administered either concomitantly, e.g., as anadmixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In another specific embodiment, compositions of the invention are usedin combination with PNEUMOVAX-23™ to treat, prevent, and/or diagnoseinfection and/or any disease, disorder, and/or condition associatedtherewith. In one embodiment, compositions of the invention are used incombination with PNEUMOVAX-23™ to treat, prevent, and/or diagnose anyGram positive bacterial infection and/or any disease, disorder, and/orcondition associated therewith. In another embodiment, compositions ofthe invention are used in combination with PNEUMOVAX-23™ to treat,prevent, and/or diagnose infection and/or any disease, disorder, and/orcondition associated with one or more members of the genus Enterococcusand/or the genus Streptococcus. In another embodiment, compositions ofthe invention are used in any combination with PNEUMOVAX-23™ to treat,prevent, and/or diagnose infection and/or any disease, disorder, and/orcondition associated with one or more members of the Group Bstreptococci. In another embodiment, compositions of the invention areused in combination with PNEUMOVAX-23™ to treat, prevent, and/ordiagnose infection and/or any disease, disorder, and/or conditionassociated with Streptococcus pneumoniae.

The compositions of the invention may be administered alone or incombination with other therapeutic agents, including but not limited to,chemotherapeutic agents, antibiotics, antivirals, steroidal andnon-steroidal anti-inflammatories, conventional immunotherapeutic agentsand cytokines. Combinations may be administered either concomitantly,e.g., as an admixture, separately but simultaneously or concurrently; orsequentially. This includes presentations in which the combined agentsare administered together as a therapeutic mixture, and also proceduresin which the combined agents are administered separately butsimultaneously, e.g., as through separate intravenous lines into thesame individual. Administration “in combination” further includes theseparate administration of one of the compounds or agents given first,followed by the second.

In a specific embodiment, the compositions of the invention areadministered in combination with antagonists of co-activators of B cells(e.g., Neutrokine-Alpha and/or Neutrokine-AlphaSV (InternationalApplication No. PCT/US96/17957), BLyS, BAFF, TALL-1, THANK, and/orzTNF4). In a further embodiment, antagonists may include, but are notlimited to, antibodies directed to and/or the soluble extracellularportion of the Neutokine-Alpha and/or Neutrokine-AlphaSV receptor, theBLyS receptor, the BAFF receptor, the TALL-1 receptor, the THANKreceptor, TACI, and/or BCMA.

In one embodiment, the compositions of the invention are administered incombination with other members of the TNF family. TNF, TNF-related orTNF-like molecules that may be administered with the compositions of theinvention include, but are not limited to, soluble forms of TNF-alpha,lymphotoxin-alpha (LT-alpha, also known as TNF-beta), LT-beta (found incomplex heterotrimer LT-alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L,4-1BBL, DcR3, OX40L, TNF-gamma (International Publication No. WO96/14328), AIM-I (International Publication No. WO 97/33899),endokine-alpha (International Publication No. WO 98/07880),Neutrokine-Alpha and/or Neutrokine-AlphaSV (International ApplicationNo. PCT/US96/17957), TR6 (International Publication No. WO 98/30694),OPG, OX40, nerve growth factor (NGF), and soluble forms of Fas, CD30,CD27, CD40 and 4-IBB, TR2 (International Publication No. WO 96/34095),DR3 (International Publication No. WO 97/33904), DR4 (InternationalPublication No. WO 98/32856), TR5 (International Publication No. WO98/30693), TR6 (International Publication No. WO 98/30694), TR7(International Publication No. WO 98/41629), TRANK, TR9 (InternationalPublication No. WO 98/56892), TR10 (International Publication No. WO98/54202), 312C2 (International Publication No. WO 98/06842), and TR12,and soluble forms CD154, CD70, and CD153.

In a preferred embodiment, the compositions of the invention areadministered in combination with CD40 ligand (CD40L), a soluble form ofCD40L (e.g., AVREND™), biologically active fragments, variants, orderivatives of CD40L, anti-CD40L antibodies (e.g., agonistic orantagonistic antibodies), and/or anti-CD40 antibodies (e.g, agonistic orantagonistic antibodies).

In another embodiment, compositions of the invention are administered incombination with an anticoagulant. Anticoagulants that may beadministered with the compositions of the invention include, but are notlimited to, heparin, warfarin, and aspirin. In a specific embodiment,compositions of the invention are administered in combination withheparin and/or warfarin. In another specific embodiment, compositions ofthe invention are administered in combination with warfarin. In anotherspecific embodiment, compositions of the invention are administered incombination with warfarin and aspirin. In another specific embodiemtn,compositions of the invention are administered in combination withheparin. In another specific embodiemtn, compositions of the inventionare administered in combination with heparin and aspirin.

In another embodiment, compositions of the invention are administered incombination with an agent that suppresses the production ofanticardiolipin antibodies. In specific embodiments, the polynucleotidesof the invention are administered in combination with an agent thatblocks and/or reduces the ability of anticardiolipin antibodies to bindphospholipid-binding plasma protein beta 2-glycoprotein I (b2GPI).

In certain embodiments, compositions of the invention are administeredin combination with antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors. Nucleoside reverse transcriptaseinhibitors that may be administered in combination with the compositionsof the invention, include, but are not limited to, RETROVIR™(zidovudine/AZT), VIDEX™ (didanosine/ddI), HIVID™ (zalcitabine/ddC),ZERIT™ (stavudine/d4T), EPIVIR™ (lamivudine/3TC), and COMBUVIR™(zidovudine/lamivudine). Non-nucleoside reverse transcriptase inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, VIRAMUNE™ (nevirapine),RESCRIPTOR™ (delavirdine), and SUSTIVA™ (efavirenz). Protease inhibitorsthat may be administered in combination with the compositions of theinvention, include, but are not limited to, CRIXIVAN™ (indinavir),NORVIR™ (ritonavir), INVRASE™ (saquinavir), and VIRACEPT™ (nelfinavir).In a specific embodiment, antiretroviral agents, nucleoside reversetranscriptase inhibitors, non-nucleoside reverse transcriptaseinhibitors, and/or protease inhibitors may be used in any combinationwith compositions of the invention to treat, prevent, and/or diagnoseAIDS and/or to treat, prevent, and/or diagnose HIV infection.

In other embodiments, compositions of the invention may be administeredin combination with anti-opportunistic infection agents.Anti-opportunistic agents that may be administered in combination withthe compositions of the invention, include, but are not limited to,TRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDINE™, ATOVAQUONE™,ISONIAZID™, RIFAMPIN™, PYRAZINAMIDE™, ETHAMBUTOL™, RIFABUTIN™,CLARITHROMYCIN™, AZITHROMYCIN™, GANCICLOVIR™, FOSCARNET™, CIDOFOVIR™,FLUCONAZOLE™, ITRACONAZOLE™, KETOCONAZOLE™, ACYCLOVIR™, FAMCICOLVIR™,PYRIMETHAMINE™, LEUCOVORIN™, NEUPOGEN™ (filgrastim/G-CSF), and LEUKINE™(sargramostim/GM-CSF). In a specific embodiment, compositions of theinvention are used in any combination withTRIMETHOPRIM-SULFAMETHOXAZOLE™, DAPSONE™, PENTAMIDNE™, and/orATOVAQUONE™ to prophylactically treat, prevent, and/or diagnose anopportunistic Pneumocystis carinii pneumonia infection. In anotherspecific embodiment, compositions of the invention are used in anycombination with ISONIAZID™, REFAMPIN™, PYRAZINAMIDE™, and/orETHAMBUTOL™ to prophylactically treat, prevent, and/or diagnose anopportunistic Mycobacterium avium complex infection. In another specificembodiment, compositions of the invention are used in any combinationwith RIFABUTIN™, CLARITHROMYCIN™, and/or AZITHROMYCIN™ toprophylactically treat, prevent, and/or diagnose an opportunisticMycobacterium tuberculosis infection. In another specific embodiment,compositions of the invention are used in any combination withGANCICLOVIR™, FOSCARNET™, and/or CIDOFOVIR™ to prophylactically treat,prevent, and/or diagnose an opportunistic cytomegalovirus infection. Inanother specific embodiment, compositions of the invention are used inany combination with FLUCONAZOLE™, ITRACONAZOLE™, and/or KETOCONAZOLE™to prophylactically treat, prevent, and/or diagnose an opportunisticfungal infection. In another specific embodiment, compositions of theinvention are used in any combination with ACYCLOVIR™ and/orFAMCICOLVIR™ to prophylactically treat, prevent, and/or diagnose anopportunistic herpes simplex virus type I and/or type II infection. Inanother specific embodiment, compositions of the invention are used inany combination with PYRIMETHAMINE™ and/or LEUCOVORIN™ toprophylactically treat, prevent, and/or diagnose an opportunisticToxoplasma gondii infection. In another specific embodiment,compositions of the invention are used in any combination withLEUCOVORIN™ and/or NEUPOGEN™ to prophylactically treat, prevent, and/ordiagnose an opportunistic bacterial infection.

In a further embodiment, the compositions of the invention areadministered in combination with an antiviral agent. Antiviral agentsthat may be administered with the compositions of the invention include,but are not limited to, acyclovir, ribavirin, amantadine, andremantidine.

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, amoxicillin, aminoglycosides, beta-lactam(glycopeptide), beta-lactamases, Clindamycin, chloramphenicol,cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin,fluoroquinolones, macrolides, metronidazole, penicillins, quinolones,rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim,trimethoprim-sulfamthoxazole, and vancomycin.

Conventional nonspecific immunosuppressive agents, that may beadministered in combination with the compositions of the inventioninclude, but are not limited to, steroids, cyclosporine, cyclosporineanalogs cyclophosphamide, cyclophosphamide IV, methylprednisolone,prednisolone, azathioprine, FK-506, 15-deoxyspergualin, and otherimmunosuppressive agents that act by suppressing the function ofresponding T cells.

In specific embodiments, compositions of the invention are administeredin combination with immunosuppressants. Immunosuppressants preparationsthat may be administered with the compositions of the invention include,but are not limited to, ORTHOCLONE™ (OKT3), SANDIMMUNE™/NEORAL™/SANGDYA™(cyclosporin), PROGRAF™ (tacrolimus), CELLCEPT™ (mycophenolate),Azathioprine, glucorticosteroids, and RAPAMUNE™ (sirolimus). In aspecific embodiment, immunosuppressants may be used to prevent rejectionof organ or bone marrow transplantation.

In a preferred embodiment, the compositions of the invention areadministered in combination with steroid therapy. Steroids that may beadministered in combination with the compositions of the invention,include, but are not limited to, oral corticosteroids, prednisone, andmethylprednisolone (e.g., IV methylprednisolone). In a specificembodiment, compositions of the invention are administered incombination with prednisone. In a further specific embodiment, thecompositions of the invention are administered in combination withprednisone and an immunosuppressive agent. Immunosuppressive agents thatmay be administered with the compositions of the invention andprednisone are those described herein, and include, but are not limitedto, azathioprine, cylophosphamide, and cyclophosphamide IV. In a anotherspecific embodiment, compositions of the invention are administered incombination with methylprednisolone. In a further specific embodiment,the compositions of the invention are administered in combination withmethylprednisolone and an immunosuppressive agent. Immunosuppressiveagents that may be administered with the compositions of the inventionand methylprednisolone are those described herein, and include, but arenot limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.

In a preferred embodiment, the compositions of the invention areadministered in combination with an antimalarial. Antimalarials that maybe administered with the compositions of the invention include, but arenot limited to, hydroxychloroquine, chloroquine, and/or quinacrine.

In a preferred embodiment, the compositions of the invention areadministered in combination with an NSAID.

In a nonexclusive embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five, ten, ormore of the following drugs: NRD-101 (Hoechst Marion Roussel),diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin(Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton(Abbott), valdecoxib (Monsanto), eltenac (Byk Gulden), campath, AGM-1470(Takeda), CDP-571 (Celltech Chiroscience), CM-101 (CarboMed), ML-3000(Merckle), CB-2431 (KS Biomedix), CBF-BS2 (KS Biomedix), IL-lRa genetherapy (Valentis), JTE-522 (Japan Tobacco), paclitaxel (Angiotech),DW-166HC (Dong Wha), darbufelone mesylate (Warner-Lambert), soluble TNFreceptor 1 (synergen; Amgen), IPR-6001 (Institute for PharmaceuticalResearch), trocade (Hoffman-La Roche), EF-5 (Scotia Pharmaceuticals),BIIL-284 (Boehringer Ingelheim), BIIF-1149 (Boehringer Ingelheim),LeukoVax (Inflammatics), MK-663 (Merck), ST-1482 (Sigma-Tau), andbutixocort propionate (WarnerLambert).

In a preferred embodiment, the compositions of the invention areadministered in combination with one, two, three, four, five or more ofthe following drugs: methotrexate, sulfasalazine, sodium aurothiomalate,auranofin, cyclosporine, penicillamine, azathioprine, an antimalarialdrug (e.g., as described herein), cyclophosphamide, chlorambucil, gold,ENBREL™ (Etanercept), anti-TNF antibody, LJP 394 (La JollaPharmaceutical Company, San Diego, Calif.), and prednisolone.

In a more preferred embodiment, the compositions of the invention areadministered in combination with an antimalarial, methotrexate, anti-TNFantibody, ENBREL™ and/or suflasalazine. In one embodiment, thecompositions of the invention are administered in combination withmethotrexate. In another embodiment, the compositions of the inventionare administered in combination with anti-TNF antibody. In anotherembodiment, the compositions of the invention are administered incombination with methotrexate and anti-TNF antibody. In anotherembodiment, the compositions of the invention are administered incombination with suflasalazine. In another specific embodiment, thecompositions of the invention are administered in combination withmethotrexate, anti-TNF antibody, and suflasalazine. In anotherembodiment, the compositions of the invention are administered incombination ENBREL™. In another embodiment, the compositions of theinvention are administered in combination with ENBREL™ and methotrexate.In another embodiment, the compositions of the invention areadministered in combination with ENBREL™, methotrexate andsuflasalazine. In another embodiment, the compositions of the inventionare administered in combination with ENBREL™, methotrexate andsuflasalazine. In other embodiments, one or more antimalarials iscombined with one of the above-recited combinations. In a specficembodiment, the compositions of the invention are administered incombination with an antimalarial (e.g., hydroxychloroquine), ENBREL™,methotrexate and suflasalazine. In another specfic embodiment, thecompositions of the invention are administered in combination with anantimalarial (e.g., hydroxychloroquine), sulfasalazine, anti-TNFantibody, and methotrexate.

In an additional embodiment, compositions of the invention areadministered alone or in combination with one or more intravenous immuneglobulin preparations. Intravenous immune globulin preparations that maybe administered with the compositions of the invention include, but notlimited to, GAMMAR™, IVEEGAM™, SANDOGLOBULIN™, GAMMAGARD S/D™, andGAMIMUNE™. In a specific embodiment, compositions of the invention areadministered in combination with intravenous immune globulinpreparations in transplantation therapy (e.g., bone marrow transplant).

In a further embodiment, the compositions of the invention areadministered in combination with an antibiotic agent. Antibiotic agentsthat may be administered with the compositions of the invention include,but are not limited to, tetracycline, metronidazole, amoxicillin,beta-lactamases, aminoglycosides, macrolides, quinolones,fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, andstreptomycin.

In an additional embodiment, the compositions of the invention areadministered alone or in combination with an anti-inflammatory agent.Anti-inflammatory agents that may be administered with the compositionsof the invention include, but are not limited to, glucocorticoids andthe nonsteroidal anti-inflammatories, aminoarylcarboxylic acidderivatives, arylacetic acid derivatives, arylbutyric acid derivatives,arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles,pyrazolones, salicylic acid derivatives, thiazinecarboxamides,e-acetamidocaproic acid, S-adenosylmethionine, 3-amino-4-hydroxybutyricacid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide,ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein,oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, andtenidap.

In another embodiment, compostions of the invention are administered incombination with a chemotherapeutic agent. Chemotherapeutic agents thatmay be administered with the compositions of the invention include, butare not limited to, antibiotic derivatives (e.g., doxorubicin,bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g.,tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate,floxuridine, interferon alpha-2b, glutamic acid, plicamycin,mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine,BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide,estramustine, hydroxyurea, procarbazine, mitomycin, busulfan,cis-platin, and vincristine sulfate); hormones (e.g.,medroxyprogesterone, estramustine phosphate sodium, ethinyl estradiol,estradiol, megestrol acetate, methyltestosterone, diethylstilbestroldiphosphate, chlorotrianisene, and testolactone); nitrogen mustardderivatives (e.g., mephalen, chorambucil, mechlorethamine (nitrogenmustard) and thiotepa); steroids and combinations (e.g., bethamethasonesodium phosphate); and others (e.g., dicarbazine, asparaginase,mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).

In a specific embodiment, compositions of the invention are administeredin combination with CHOP (cyclophosphamide, doxorubicin, vincristine,and prednisone) or any combination of the components of CHOP. In anotherembodiment, compositions of the invention are administered incombination with Rituximab. In a further embodiment, compositions of theinvention are administered with Rituxmab and CHOP, or Rituxmab and anycombination of the components of CHOP.

In an additional embodiment, the compositions of the invention areadministered in combination with cytokines. Cytokines that may beadministered with the compositions of the invention include, but are notlimited to, GM-CSF, G-CSF, IL2, IL3, ILA, IL5, IL6, IL7, IL10, IL12,IL13, IL15, anti-CD40, CD40L, IFN-alpha, IFN-beta, IFN-gamma, TNF-alpha,and TNF-beta. In another embodiment, compositions of the invention maybe administered with any interleukin, including, but not limited to,IL-1 alpha, IL-1beta, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9,IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19,IL-20, IL-21, and IL-22. In preferred embodiments, the compositions ofthe invention are administered in combination with IL4 and IL10. BothIL4 and IL10 have been observed by the inventors to enhanceD-SLAM-mediated B cell proliferation.

In an additional embodiment, the compositions of the invention areadministered with a chemokine. In another embodiment, the compositionsof the invention are administered with chemokine beta-8, chemokinebeta-1, and/or macrophage inflammatory protein-4. In a preferredembodiment, the compositions of the invention are administered withchemokine beta-8.

In an additional embodiment, the compositions of the invention areadministered in combination with an IL-4 antagonist. IL-4 antagoniststhat may be administered with the compositions of the invention include,but are not limited to: soluble IL-4 receptor polypeptides, multimericforms of soluble IL-4 receptor polypeptides; anti-IL-4 receptorantibodies that bind the IL-4 receptor without transducing thebiological signal elicited by IL-4, anti-IL4 antibodies that blockbinding of IL-4 to one or more IL-4 receptors, and muteins of IL-4 thatbind IL-4 receptors but do not transduce the biological signal elicitedby IL-4. Preferably, the antibodies employed according to this methodare monoclonal antibodies (including antibody fragments, such as, forexample, those described herein).

In an additional embodiment, the compositions of the invention areadministered in combination with hematopoietic growth factors.Hematopoietic growth factors that may be administered with thecompositions of the invention include, but are not limited to, LEUKINE™(SARGRAMOSTIM™) and NEUPOGEN™ (FILGRASTIM™).

In an additional embodiment, the compositions of the invention areadministered in combination with angiogenic proteins. Angiogenicproteins that may be administered with the compositions of the inventioninclude, but are not limited to, Glioma Derived Growth Factor (GDGF), asdisclosed in European Patent Number EP-399816; Platelet Derived GrowthFactor-A (PDGF-A), as disclosed in European Patent Number EP-682110;Platelet Derived Growth Factor-B (PDGF-B), as disclosed in EuropeanPatent Number EP-282317; Placental Growth Factor (PlGF), as disclosed inInternational Publication Number WO 92/06194; Placental Growth Factor-2(PlGF-2), as disclosed in Hauser et al., Growth Factors, 4:259-268(1993); Vascular Endothelial Growth Factor (VEGF), as disclosed inInternational Publication Number WO 90/13649; Vascular EndothelialGrowth Factor-A (VEGF-A), as disclosed in European Patent NumberEP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosedin International Publication Number WO 96/39515; Vascular EndothelialGrowth Factor B-186 (VEGF-B186), as disclosed in InternationalPublication Number WO 96/26736; Vascular Endothelial Growth Factor-D(VEGF-D), as disclosed in International Publication Number WO 98/02543;Vascular Endothelial Growth Factor-D (VEGF-D), as disclosed inInternational Publication Number WO 98/07832; and Vascular EndothelialGrowth Factor-E (VEGF-E), as disclosed in German Patent NumberDE19639601. The above mentioned references are incorporated herein byreference herein.

In an additional embodiment, the compositions of the invention areadministered in combination with Fibroblast Growth Factors. FibroblastGrowth Factors that may be administered with the compositions of theinvention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4,FGF-5, FGF-6, FGF-7, FGF-8, FGF-9, FGF-10, FGF-11, FGF-12, FGF-13,FGF-14, and FGF-15.

In additional embodiments, the compositions of the invention areadministered alone or in combination with other therapeutic orprophylactic regimens, including but not limited to, radiation therapy.Such combinatorial therapy may be administered sequentially and/orconcomitantly.

Example 25 Method of Treating Decreased Levels of D-SLAM

The present invention relates to a method for treating an individual inneed of a decreased level of D-SLAM activity in the body comprising,administering to such an individual a composition comprising atherapeutically effective amount of D-SLAM antagonist. Preferredantagonists for use in the present invention are D-SLAM-specificantibodies.

Moreover, it will be appreciated that conditions caused by a decrease inthe standard or normal expression level of D-SLAM in an individual canbe treated by administering D-SLAM, preferably in the secreted form.Thus, the invention also provides a method of treatment of an individualin need of an increased level of D-SLAM polypeptide comprisingadministering to such an individual a pharmaceutical compositioncomprising an amount of D-SLAM to increase the activity level of D-SLAMin such an individual.

For example, a patient with decreased levels of D-SLAM polypeptidereceives a daily dose 0.1-100 ug/kg of the polypeptide for sixconsecutive days. Preferably, the polypeptide is in the secreted form.The exact details of the dosing scheme, based on administration andformulation, are provided in Example 24.

Example 26 Method of Treating Increased Levels of D-SLAM

The present invention also relates to a method for treating anindividual in need of an increased level of D-SLAM activity in the bodycomprising administering to such an individual a composition comprisinga therapeutically effective amount of D-SLAM or an agonist thereof.

Antisense technology is used to inhibit production of D-SLAM. Thistechnology is one example of a method of decreasing levels of D-SLAMpolypeptide, preferably a secreted form, due to a variety of etiologies,such as cancer.

For example, a patient diagnosed with abnormally increased levels ofD-SLAM is administered intravenously antisense polynucleotides at 0.5,1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeatedafter a 7-day rest period if the treatment was well tolerated. Theformulation of the antisense polynucleotide is provided in Example 24.

Example 27 Method of Treatment Using Gene Therapy—Ex Vivo

One method of gene therapy transplants fibroblasts, which are capable ofexpressing D-SLAM polypeptides, onto a patient. Generally, fibroblastsare obtained from a subject by skin biopsy. The resulting tissue isplaced in tissue-culture medium and separated into small pieces. Smallchunks of the tissue are placed on a wet surface of a tissue cultureflask, approximately ten pieces are placed in each flask. The flask isturned upside down, closed tight and left at room temperature overnight. After 24 hours at room temperature, the flask is inverted and thechunks of tissue remain fixed to the bottom of the flask and fresh media(e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) isadded. The flasks are then incubated at 37° C. for approximately oneweek.

At this time, fresh media is added and subsequently changed everyseveral days. After an additional two weeks in culture, a monolayer offibroblasts emerge. The monolayer is trypsinized and scaled into largerflasks.

pMV-7 (Kirschmeier, P. T. et al., DNA, 7:219-25 (1988)), flanked by thelong terminal repeats of the Moloney murine sarcoma virus, is digestedwith EcoRI and HindIII and subsequently treated with calf intestinalphosphatase. The linear vector is fractionated on agarose gel andpurified, using glass beads.

The cDNA encoding D-SLAM can be amplified using PCR primers whichcorrespond to the 5′ and 3′ end sequences respectively as set forth inExample 1. Preferably, the 5′ primer contains an EcoRI site and the 3′primer includes a HindIII site. Equal quantities of the Moloney murinesarcoma virus linear backbone and the amplified EcoRI and HindIIIfragment are added together, in the presence of T4 DNA ligase. Theresulting mixture is maintained under conditions appropriate forligation of the two fragments. The ligation mixture is then used totransform bacteria HB101, which are then plated onto agar containingkanamycin for the purpose of confirming that the vector containsproperly inserted D-SLAM.

The amphotropic pA317 or GP+am12 packaging cells are grown in tissueculture to confluent density in Dulbecco's Modified Eagles Medium (DMEM)with 10% calf serum (CS), penicillin and streptomycin. The MSV vectorcontaining the D-SLAM gene is then added to the media and the packagingcells transduced with the vector. The packaging cells now produceinfectious viral particles containing the D-SLAM gene (the packagingcells are now referred to as producer cells).

Fresh media is added to the transduced producer cells, and subsequently,the media is harvested from a 10 cm plate of confluent producer cells.The spent media, containing the infectious viral particles, is filteredthrough a millipore filter to remove detached producer cells and thismedia is then used to infect fibroblast cells. Media is removed from asub-confluent plate of fibroblasts and quickly replaced with the mediafrom the producer cells. This media is removed and replaced with freshmedia. If the titer of virus is high, then virtually all fibroblastswill be infected and no selection is required. If the titer is very low,then it is necessary to use a retroviral vector that has a selectablemarker, such as neo or his. Once the fibroblasts have been efficientlyinfected, the fibroblasts are analyzed to determine whether D-SLAMprotein is produced.

The engineered fibroblasts are then transplanted onto the host, eitheralone or after having been grown to confluence on cytodex 3 microcarrierbeads.

Example 28 Gene Therapy Using Endogenous D-SLAM Gene

Another method of gene therapy according to the present inventioninvolves operably associating the endogenous D-SLAM sequence with apromoter via homologous recombination as described, for example, in U.S.Pat. No. 5,641,670, issued Jun. 24, 1997; International Publication No.WO 96/29411, published Sep. 26, 1996; International Publication No. WO94/12650, published Aug. 4, 1994; Koller et al., Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); and Zijlstra et al., Nature 342:435-438 (1989).This method involves the activation of a gene which is present in thetarget cells, but which is not expressed in the cells, or is expressedat a lower level than desired.

Polynucleotide constructs are made which contain a promoter andtargeting sequences, which are homologous to the 5′ non-coding sequenceof endogenous D-SLAM, flanking the promoter. The targeting sequence willbe sufficiently near the 5′ end of D-SLAM so the promoter will beoperably linked to the endogenous sequence upon homologousrecombination. The promoter and the targeting sequences can be amplifiedusing PCR. Preferably, the amplified promoter contains distinctrestriction enzyme sites on the 5′ and 3′ ends. Preferably, the 3′ endof the first targeting sequence contains the same restriction enzymesite as the 5′ end of the amplified promoter and the 5′ end of thesecond targeting sequence contains the same restriction site as the 3′end of the amplified promoter.

The amplified promoter and the amplified targeting sequences aredigested with the appropriate restriction enzymes and subsequentlytreated with calf intestinal phosphatase. The digested promoter anddigested targeting sequences are added together in the presence of T4DNA ligase. The resulting mixture is maintained under conditionsappropriate for ligation of the two fragments. The construct is sizefractionated on an agarose gel then purified by phenol extraction andethanol precipitation.

In this Example, the polynucleotide constructs are administered as nakedpolynucleotides via electroporation. However, the polynucleotideconstructs may also be administered with transfection-facilitatingagents, such as liposomes, viral sequences, viral particles,precipitating agents, etc. Such methods of delivery are known in theart.

Once the cells are transfected, homologous recombination will take placewhich results in the promoter being operably linked to the endogenousD-SLAM sequence. This results in the expression of D-SLAM in the cell.Expression may be detected by immunological staining, or any othermethod known in the art.

Fibroblasts are obtained from a subject by skin biopsy. The resultingtissue is placed in DMEM+10% fetal calf serum. Exponentially growing orearly stationary phase fibroblasts are trypsinized and rinsed from theplastic surface with nutrient medium. An aliquot of the cell suspensionis removed for counting, and the remaining cells are subjected tocentrifugation. The supernatant is aspirated and the pellet isresuspended in 5 ml of electroporation buffer (20 mM HEPES pH 7.3, 137mM NaCl, 5 mM KCl, 0.7 mM Na₂ HPO₄, 6 mM dextrose). The cells arerecentrifuged, the supernatant aspirated, and the cells resuspended inelectroporation buffer containing 1 mg/ml acetylated bovine serumalbumin. The final cell suspension contains approximately 3×10⁶cells/ml. Electroporation should be performed immediately followingresuspension.

Plasmid DNA is prepared according to standard techniques. For example,to construct a plasmid for targeting to the D-SLAM locus, plasmid pUC18(MBI Fermentas, Amherst, N.Y.) is digested with HindIII. The CMVpromoter is amplified by PCR with an XbaI site on the 5′ end and a BamHIsite on the 3′end. Two D-SLAM non-coding sequences are amplified viaPCR: one D-SLAM non-coding sequence (D-SLAM fragment 1) is amplifiedwith a HindIII site at the 5′ end and an Xba site at the 3′end; theother D-SLAM non-coding sequence (D-SLAM fragment 2) is amplified with aBamHI site at the 5′end and a HindIII site at the 3′end. The CMVpromoter and D-SLAM fragments are digested with the appropriate enzymes(CMV promoter—XbaI and BamHI; D-SLAM fragment 1—XbaI; D-SLAM fragment2—BamHI) and ligated together. The resulting ligation product isdigested with HindIII, and ligated with the HindIII-digested pUC18plasmid.

Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap(Bio-Rad). The final DNA concentration is generally at least 120 μg/ml.0.5 ml of the cell suspension (containing approximately 1.5×10⁶ cells)is then added to the cuvette, and the cell suspension and DNA solutionsare gently mixed. Electroporation is performed with a Gene-Pulserapparatus (Bio-Rad). Capacitance and voltage are set at 960 μF and250-300 V, respectively. As voltage increases, cell survival decreases,but the percentage of surviving cells that stably incorporate theintroduced DNA into their genome increases dramatically. Given theseparameters, a pulse time of approximately 14-20 mSec should be observed.

Electroporated cells are maintained at room temperature forapproximately 5 min, and the contents of the cuvette are then gentlyremoved with a sterile transfer pipette. The cells are added directly to10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cmdish and incubated at 37° C. The following day, the media is aspiratedand replaced with 10 ml of fresh media and incubated for a further 16-24hours.

The engineered fibroblasts are then injected into the host, either aloneor after having been grown to confluence on cytodex 3 microcarrierbeads. The fibroblasts now produce the protein product. The fibroblastscan then be introduced into a patient as described above.

Example 29 Method of Treatment Using Gene Therapy—In Vivo

Another aspect of the present invention is using in vivo gene therapymethods to treat, diagnose, detect, and/or prevent disorders, diseasesand conditions. The gene therapy method relates to the introduction ofnaked nucleic acid (DNA, RNA, and antisense DNA or RNA) D-SLAM sequencesinto an animal to increase or decrease the expression of the D-SLAMpolypeptide. The D-SLAM polynucleotide may be operatively linked to apromoter or any other genetic elements necessary for the expression ofthe D-SLAM polypeptide by the target tissue. Such gene therapy anddelivery techniques and methods are known in the art, see, for example,WO 90/11092, WO 98/11779; U.S. Pat. Nos. 5,693,622, 5,705,151,5,580,859; Tabata H. et al. (1997) Cardiovasc. Res. 35(3):470-479, ChaoJ et al. (1997) Pharmacol. Res. 35(6):517-522, Wolff J. A. (1997)Neuromuscul. Disord. 7(5):314-318, Schwartz B. et al. (1996) Gene Ther.3(5):405-411, Tsurumi Y. et al. (1996) Circulation 94(12):3281-3290(incorporated herein by reference).

The D-SLAM polynucleotide constructs may be delivered by any method thatdelivers injectable materials to the cells of an animal, such as,injection into the interstitial space of tissues (heart, muscle, skin,lung, liver, intestine and the like). The D-SLAM polynucleotideconstructs can be delivered in a pharmaceutically acceptable liquid oraqueous carrier.

The term “naked” polynucleotide, DNA or RNA, refers to sequences thatare free from any delivery vehicle that acts to assist, promote, orfacilitate entry into the cell, including viral sequences, viralparticles, liposome formulations, lipofectin or precipitating agents andthe like. However, the D-SLAM polynucleotides may also be delivered inliposome formulations (such as those taught in Felgner P. L. et al.(1995) Ann. NY Acad. Sci. 772:126-139 and Abdallah B. et al. (1995)Biol. Cell 85(1):1-7) which can be prepared by methods well known tothose skilled in the art.

The D-SLAM polynucleotide vector constructs used in the gene therapymethod are preferably constructs that will not integrate into the hostgenome nor will they contain sequences that allow for replication. Anystrong promoter known to those skilled in the art can be used fordriving the expression of DNA. Unlike other gene therapies techniques,one major advantage of introducing naked nucleic acid sequences intotarget cells is the transitory nature of the polynucleotide synthesis inthe cells. Studies have shown that non-replicating DNA sequences can beintroduced into cells to provide production of the desired polypeptidefor periods of up to six months.

The D-SLAM polynucleotide construct can be delivered to the interstitialspace of tissues within the an animal, including of muscle, skin, brain,lung, liver, spleen, bone marrow, thymus, heart, lymph, blood, bone,cartilage, pancreas, kidney, gall bladder, stomach, intestine, testis,ovary, uterus, rectum, nervous system, eye, gland, and connectivetissue. Interstitial space of the tissues comprises the intercellularfluid, mucopolysaccharide matrix among the reticular fibers of organtissues, elastic fibers in the walls of vessels or chambers, collagenfibers of fibrous tissues, or that same matrix within connective tissueensheathing muscle cells or in the lacunae of bone. It is similarly thespace occupied by the plasma of the circulation and the lymph fluid ofthe lymphatic channels. Delivery to the interstitial space of muscletissue is preferred for the reasons discussed below. They may beconveniently delivered by injection into the tissues comprising thesecells. They are preferably delivered to and expressed in persistent,non-dividing cells which are differentiated, although delivery andexpression may be achieved in non-differentiated or less completelydifferentiated cells, such as, for example, stem cells of blood or skinfibroblasts. In vivo muscle cells are particularly competent in theirability to take up and express polynucleotides.

For the naked D-SLAM polynucleotide injection, an effective dosageamount of DNA or RNA will be in the range of from about 0.05 g/kg bodyweight to about 50 mg/kg body weight. Preferably the dosage will be fromabout 0.005 mg/kg to about 20 mg/kg and more preferably from about 0.05mg/kg to about 5 mg/kg. Of course, as the artisan of ordinary skill willappreciate, this dosage will vary according to the tissue site ofinjection. The appropriate and effective dosage of nucleic acid sequencecan readily be determined by those of ordinary skill in the art and maydepend on the condition being treated and the route of administration.The preferred route of administration is by the parenteral route ofinjection into the interstitial space of tissues. However, otherparenteral routes may also be used, such as, inhalation of an aerosolformulation particularly for delivery to lungs or bronchial tissues,throat or mucous membranes of the nose. In addition, naked D-SLAMpolynucleotide constructs can be delivered to arteries duringangioplasty by the catheter used in the procedure.

The dose response effects of injected D-SLAM polynucleotide in muscle invivo is determined as follows. Suitable D-SLAM template DNA forproduction of mRNA coding for D-SLAM polypeptide is prepared inaccordance with a standard recombinant DNA methodology. The templateDNA, which may be either circular or linear, is either used as naked DNAor complexed with liposomes. The quadriceps muscles of mice are theninjected with various amounts of the template DNA.

Five to six week old female and male Balb/C mice are anesthetized byintraperitoneal injection with 0.3 ml of 2.5% Avertin. A 1.5 cm incisionis made on the anterior thigh, and the quadriceps muscle is directlyvisualized. The D-SLAM template DNA is injected in 0.1 ml of carrier ina 1 cc syringe through a 27 gauge needle over one minute, approximately0.5 cm from the distal insertion site of the muscle into the knee andabout 0.2 cm deep. A suture is placed over the injection site for futurelocalization, and the skin is closed with stainless steel clips.

After an appropriate incubation time (e.g., 7 days) muscle extracts areprepared by excising the entire quadriceps. Every fifth 15 umcross-section of the individual quadriceps muscles is histochemicallystained for D-SLAM protein expression. A time course for D-SLAM proteinexpression may be done in a similar fashion except that quadriceps fromdifferent mice are harvested at different times. Persistence of D-SLAMDNA in muscle following injection may be determined by Southern blotanalysis after preparing total cellular DNA and HIRT supernatants frominjected and control mice. The results of the above experimentation inmice can be use to extrapolate proper dosages and other treatmentparameters in humans and other animals using D-SLAM naked DNA.

Example 30 D-SLAM Transgenic Animals

The D-SLAM polypeptides can also be expressed in transgenic animals.Animals of any species, including, but not limited to, mice, rats,rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows andnon-human primates, e.g., baboons, monkeys, and chimpanzees may be usedto generate transgenic animals. In a specific embodiment, techniquesdescribed herein or otherwise known in the art, are used to expresspolypeptides of the invention in humans, as part of a gene therapyprotocol.

Any technique known in the art may be used to introduce the transgene(i.e., polynucleotides of the invention) into animals to produce thefounder lines of transgenic animals. Such techniques include, but arenot limited to, pronuclear microinjection (Paterson et al., Appl.Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology(NY) 11:1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834(1991); and Hoppe et al., U.S. Pat. No. 4,873,191 (1989)); retrovirusmediated gene transfer into germ lines (Van der Putten et al., Proc.Natl. Acad. Sci., USA 82:6148-6152 (1985)), blastocysts or embryos; genetargeting in embryonic stem cells (Thompson et al., Cell 56:313-321(1989)); electroporation of cells or embryos (Lo, 1983, Mol Cell. Biol.3:1803-1814 (1983)); introduction of the polynucleotides of theinvention using a gene gun (see, e.g., Ulmer et al., Science 259:1745(1993); introducing nucleic acid constructs into embryonic pleuripotentstem cells and transferring the stem cells back into the blastocyst; andsperm-mediated gene transfer (Lavitrano et al., Cell 57:717-723 (1989);etc. For a review of such techniques, see Gordon, “Transgenic Animals,”Intl. Rev. Cytol. 115:171-229 (1989), which is incorporated by referenceherein in its entirety.

Any technique known in the art may be used to produce transgenic clonescontaining polynucleotides of the invention, for example, nucleartransfer into enucleated oocytes of nuclei from cultured embryonic,fetal, or adult cells induced to quiescence (Campell et al., Nature380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)).

The present invention provides for transgenic animals that carry thetransgene in all their cells, as well as animals which carry thetransgene in some, but not all their cells, i.e., mosaic animals orchimeric. The transgene may be integrated as a single transgene or asmultiple copies such as in concatamers, e.g., head-to-head tandems orhead-to-tail tandems. The transgene may also be selectively introducedinto and activated in a particular cell type by following, for example,the teaching of Lasko et al. (Lasko et al., Proc. Natl. Acad. Sci. USA89:6232-6236 (1992)). The regulatory sequences required for such acell-type specific activation will depend upon the particular cell typeof interest, and will be apparent to those of skill in the art. When itis desired that the polynucleotide transgene be integrated into thechromosomal site of the endogenous gene, gene targeting is preferred.

Briefly, when such a technique is to be utilized, vectors containingsome nucleotide sequences homologous to the endogenous gene are designedfor the purpose of integrating, via homologous recombination withchromosomal sequences, into and disrupting the function of thenucleotide sequence of the endogenous gene. The transgene may also beselectively introduced into a particular cell type, thus inactivatingthe endogenous gene in only that cell type, by following, for example,the teaching of Gu et al. (Gu et al., Science 265:103-106 (1994)). Theregulatory sequences required for such a cell-type specific inactivationwill depend upon the particular cell type of interest, and will beapparent to those of skill in the art. The contents of each of thedocuments recited in this paragraph is herein incorporated by referencein its entirety.

Any of the D-SLAM polypeptides disclose throughout this application canbe used to generate transgenic animals. For example, DNA encoding aminoacids M1-K232 of SEQ ID NO:2 can be inserted into a vector containing apromoter, such as the actin promoter, which will ubiquitously expressthe inserted fragment. Primers that can be used to generate suchfragments include a 5′ primer containing a BamHI restriction site shownin bold: GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ ID NO:22) and a 3′ primer, containing a Xba restriction site shown in bold:GCAGCATCTAGATTATTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO: 24). Thisconstruct will express the predicted extracellular domain of D-SLAMunder the control of the actin promoter for ubiquitous expression. Theregion of D-SLAM included in this construct extends from M1-K232 of SEQID NO:2.

Similarly, the DNA encoding the full length D-SLAM protein can also beinserted into a vector using the following primers: A 5′ primercontaining a BamHI restriction site shown in bold:GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ ID NO: 22) anda 3′ primer, containing a Xba restriction site shown in bold:GCAGCATCTAGATTATGGCAGATCCTGCACAAGGGGGTTCTCTGTC (SEQ ID NO: 23). Besidesthese two examples, other fragments of D-SLAM can also be inserted intoa vector to create transgenics having ubiquitous expression.

Alternatively, polynucleotides of the invention can be inserted in avector which controls tissue specific expression through a tissuespecific promoter. For example, a construct having a transferrinpromoter would express the D-SLAM polypeptide in the liver of transgenicanimals. Therefore, DNA encoding amino acids M1-K232 of SEQ ID NO:2 canbe amplified using a 5′ primer, having a BamHI restriction site shown inbold: GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ ID NO:22), and a 3′ primer, containing a Xba restriction site shown in bold:GCAGCATCTAGATTATTTGTAGGAGGCCTTCCCTGGTGCTGCCTC (SEQ ID NO: 24).

Similarly, the DNA encoding the full length D-SLAM protein can also beinserted into a vector for tissue specific expression using thefollowing primers: A 5′ primer containing a BamHI restriction site shownin bold: GCAGCAGGATCCGCCATCATGGTCATGAGGCCCCTGTGGAGTCTGCTTCTC (SEQ ID NO:22) and a 3′ primer, containing a Xba restriction site shown in bold:GCAGCATCTAGATTATGGCAGATCCTGCACAAGGGGGTTCTCTGTC (SEQ ID NO: 23).

Once transgenic animals have been generated, the expression of therecombinant gene may be assayed utilizing standard techniques. Initialscreening may be accomplished by Southern blot analysis or PCRtechniques to analyze animal tissues to verify that integration of thetransgene has taken place. The level of mRNA expression of the transgenein the tissues of the transgenic animals may also be assessed usingtechniques which include, but are not limited to, Northern blot analysisof tissue samples obtained from the animal, in situ hybridizationanalysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenicgene-expressing tissue may also be evaluated immunocytochemically orimmunohistochemically using antibodies specific for the transgeneproduct.

Once the founder animals are produced, they may be bred, inbred,outbred, or crossbred to produce colonies of the particular animal.Examples of such breeding strategies include, but are not limited to:outbreeding of founder animals with more than one integration site inorder to establish separate lines; inbreeding of separate lines in orderto produce compound transgenics that express the transgene at higherlevels because of the effects of additive expression of each transgene;crossing of heterozygous transgenic animals to produce animalshomozygous for a given integration site in order to both augmentexpression and eliminate the need for screening of animals by DNAanalysis; crossing of separate homozygous lines to produce compoundheterozygous or homozygous lines; and breeding to place the transgene ona distinct background that is appropriate for an experimental model ofinterest.

Transgenic animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of D-SLAM polypeptides, studying conditions and/or disordersassociated with aberrant D-SLAM expression, and in screening forcompounds effective in ameliorating such conditions and/or disorders.

Example 31 D-SLAM Knock-Out Animals

Endogenous D-SLAM gene expression can also be reduced by inactivating or“knocking out” the D-SLAM gene and/or its promoter using targetedhomologous recombination. (E.g., see Smithies et al., Nature 317:230-234(1985); Thomas & Capecchi, Cell 51:503-512 (1987); Thompson et al., Cell5:313-321 (1989); each of which is incorporated by reference herein inits entirety). For example, a mutant, non-functional polynucleotide ofthe invention (or a completely unrelated DNA sequence) flanked by DNAhomologous to the endogenous polynucleotide sequence (either the codingregions or regulatory regions of the gene) can be used, with or withouta selectable marker and/or a negative selectable marker, to transfectcells that express polypeptides of the invention in vivo. In anotherembodiment, techniques known in the art are used to generate knockoutsin cells that contain, but do not express the gene of interest.Insertion of the DNA construct, via targeted homologous recombination,results in inactivation of the targeted gene. Such approaches areparticularly suited in research and agricultural fields wheremodifications to embryonic stem cells can be used to generate animaloffspring with an inactive targeted gene (e.g., see Thomas & Capecchi1987 and Thompson 1989, supra). However this approach can be routinelyadapted for use in humans provided the recombinant DNA constructs aredirectly administered or targeted to the required site in vivo usingappropriate viral vectors that will be apparent to those of skill in theart.

For example, a targeting vector can be created which incorporatesfragments of the murine D-SLAM locus flanking a Neomyocin cassette. Thetwo flanking regions are generated using the following primers. For the5′ ARM flanking region:

-   -   5′ ARM primer F: 5′AGCCGGTACCTCCGATGTGCATAATCAGGCT 3′ (SEQ ID        NO:27), with an Asp718 restriction site underlined; and    -   5′ ARM primer R: 5′ GCTTGGCGCGCCCCTGGTTAGTCTGCCTATGTA 3′ (SEQ ID        NO:28) with a BssH II restriction site underlined. For the 3′        ARM flanking region:    -   3′ ARM primer F: 5′ATCCATCGATCCACCAAGATTCACAGGCTTG 3′ (SEQ ID        NO:29) with a Cla I restriction site underlined; and    -   3′ ARM primer R: 5′ATAAGAATGCGGCCGCAAGGGCTATGCAAGCTTGGCT 3′ (SEQ        ID NO:30) with a Not I restriction site underlined.

In further embodiments of the invention, cells that are geneticallyengineered to express the polypeptides of the invention, oralternatively, that are genetically engineered not to express thepolypeptides of the invention (e.g., knockouts) are administered to apatient in vivo. Such cells may be obtained from the patient (i.e.,animal, including human) or an MHC compatible donor and can include, butare not limited to fibroblasts, bone marrow cells, blood cells (e.g.,lymphocytes), adipocytes, muscle cells, endothelial cells etc. The cellsare genetically engineered in vitro using recombinant DNA techniques tointroduce the coding sequence of polypeptides of the invention into thecells, or alternatively, to disrupt the coding sequence and/orendogenous regulatory sequence associated with the polypeptides of theinvention, e.g., by transduction (using viral vectors, and preferablyvectors that integrate the transgene into the cell genome) ortransfection procedures, including, but not limited to, the use ofplasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc. Thecoding sequence of the polypeptides of the invention can be placed underthe control of a strong constitutive or inducible promoter orpromoter/enhancer to achieve expression, and preferably secretion, ofthe D-SLAM polypeptides. The engineered cells which express andpreferably secrete the polypeptides of the invention can be introducedinto the patient systemically, e.g., in the circulation, orintraperitoneally.

Alternatively, the cells can be incorporated into a matrix and implantedin the body, e.g., genetically engineered fibroblasts can be implantedas part of a skin graft; genetically engineered endothelial cells can beimplanted as part of a lymphatic or vascular graft. (See, for example,Anderson et al. U.S. Pat. No. 5,399,349; and Mulligan & Wilson, U.S.Pat. No. 5,460,959 each of which is incorporated by reference herein inits entirety).

When the cells to be administered are non-autologous or non-MHCcompatible cells, they can be administered using well known techniqueswhich prevent the development of a host immune response against theintroduced cells. For example, the cells may be introduced in anencapsulated form which, while allowing for an exchange of componentswith the immediate extracellular environment, does not allow theintroduced cells to be recognized by the host immune system.

Knock-out animals of the invention have uses which include, but are notlimited to, animal model systems useful in elaborating the biologicalfunction of D-SLAM polypeptides, studying conditions and/or disordersassociated with aberrant D-SLAM expression, and in screening forcompounds effective in ameliorating such conditions and/or disorders.

Example 32 Assays Detecting Stimulation or Inhibition of B CellProliferation and Differentiation

Generation of functional humoral immune responses requires both solubleand cognate signaling between B-lineage cells and theirmicroenvironment. Signals may impart a positive stimulus that allows aB-lineage cell to continue its programmed development, or a negativestimulus that instructs the cell to arrest its current developmentalpathway. To date, numerous stimulatory and inhibitory signals have beenfound to influence B cell responsiveness including IL-2, IL-4, IL-5,IL-6, IL-7, IL10, IL-13, IL-14 and IL-15. Interestingly, these signalsare by themselves weak effectors but can, in combination with variousco-stimulatory proteins, induce activation, proliferation,differentiation, homing, tolerance and death among B cell populations.

One of the best studied classes of B-cell co-stimulatory proteins is theTNF-superfamily. Within this family CD40, CD27, and CD30 along withtheir respective ligands CD154, CD70, and CD153 have been found toregulate a variety of immune responses. Dendritic cells are known todirectly interact with B cells and regulate mature B cell responses atvarious stages of their differentiation. Thus, D-SLAM polypeptides ofthe invention were tested for their ability to affect B cellproliferation. Assays which allow for the detection and/or observationof the proliferation and differentiation of these B-cell populations andtheir precursors are valuable tools in determining the effects variousproteins may have on these B-cell populations in terms of proliferationand differentiation. Listed below are two assays designed to allow forthe detection of the differentiation, proliferation, or inhibition ofB-cell populations and their precursors.

In Vitro Assay—Purified D-SLAM protein, or truncated forms thereof, isassessed for its ability to induce activation, proliferation,differentiation or inhibition and/or death in B-cell populations andtheir precursors. Specifically, purified recombinant D-SLAM was assessedfor its ability to inhibit proliferation of B cells in a standardco-stimulatory assay in which purified tonsillar B cells are cultured inthe presence of anti-human IgM or pansorbin as priming agents(Sieckmann, D. G., et al., J. Exp. Med. 147:814-29 (1978); Ringden, O.,et al., Scand. J. Immunol. 6:1159-69 (1977)). The activity of D-SLAMprotein on purified human tonsillar B cells, measured qualitatively overthe dose range from 0.1 to 10,000 ng/mL, is assessed in a standardB-lymphocyte co-stimulation assay in which purified tonsillar B cellsare cultured in the presence of either formalin-fixed Staphylococcusaureus Cowan I (SAC) or immobilized anti-human IgM antibody as thepriming agent. Second signals such as IL-2 and IL-15 synergize with SAC(Pansorbin) and IgM crosslinking to elicit B cell proliferation asmeasured by tritiated-thymidine incorporation. Novel synergizing agentscan be readily identified using this assay. The assay involves isolatinghuman tonsillar B cells by magnetic bead (MACS) depletion ofCD3-positive cells. The resulting cell population is greater than 95% Bcells as assessed by expression of CD45R(B220).

Various dilutions of each sample are placed into individual wells of a96-well plate to which are added 10⁻⁵ B-cells suspended in culturemedium (RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin,10 ug/ml streptomycin, and 10⁻⁵ dilution of SAC) in a total volume of150 ul. Proliferation or inhibition is quantitated by a 20 h pulse (1uCi/well) with 3H-thymidine (6.7 Ci/mM) beginning 72 h post factoraddition. The positive and negative controls are IL2 and mediumrespectively.

B lymphocyte proliferation assay of D-SLAM. Human tonsillar B cells werepurified by magnetic bead (MACS) depletion of CD3-positive cells. Theresulting cell population was routinely greater than 95% B cells asassessed by expression of CD19 and CD20. Various dilutions of humanrecombinant D-SLAM or D-SLAM.Fc (M1-D233 of SEQ ID NO:2 fused to the Fcportion) were placed into individual wells of a 96-well plate to whichwas added 10⁵ B cells suspended in culture medium (RPMI 1640 containing10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin, 100 microgram/mlstreptomycin, and 10⁻⁵ dilution of Pansorbin (SAC) or anti-IgM) in atotal volume of 150 microliters. Proliferation was quantitated by a 20 hpulse (1 microCi/well) of ³H-thymidine (6.7 Ci/mM) beginning 72 h postfactor addition.

Results: Treatment of B cell cultures stimulated to proliferate witheither anti-human IgM or with pansorbin with recombinant D-SLAM orD-SLAM.Fc resulted in a dose-dependent inhibition of tonsillar B cellproliferation. A dose-dependent response is readily observed as theamount of crosslinking agent increases in the presence of a fixedconcentration of recombinant D-SLAM. Under the same assay conditions,TR9.Fc, an unrelated Fc fusion protein, demonstrated no inhibition.

D-SLAM agonists of the invention may also be examined in this Blymphocyte proliferation assay. In addition, one skilled in the artcould easily modify the exemplified studies to test the activity ofD-SLAM polynucleotides (e.g., gene therapy) and/or antagonists ofD-SLAM.

Antagonists (e.g., including, but not limited to, D-SLAM polypeptidefragments, antibodies directed to D-SLAM or fragments thereof) accordingto the invention exhibit a increased B cell proliferation when comparedto controls containing the same number of B cells and the sameconcentration of priming agent, and the same concentration of a solubleform of an agent that elicits an increase in B cell proliferativeactivity in the absence the antagonist. Agonists (e.g., including butnot limited to antibodies directed to D-SLAM, or fragments thereof)demonstrate an decreased B cell proliferation when compared to thatobserved when the same number of B cells is contacted with the sameconcentration of priming agent.

In Vivo Assay—BALB/c mice are injected (i.p.) twice per day with bufferonly, or 2 mg/Kg of D-SLAM protein, or truncated forms thereof. Micereceive this treatment for 4 consecutive days, at which time they aresacrificed and various tissues and serum collected for analyses.Comparison of H&E sections from normal and D-SLAM protein-treatedspleens identify the results of the activity of D-SLAM protein on spleencells, such as the diffusion of peri-arterial lymphatic sheaths, and/orsignificant increases in the nucleated cellularity of the red pulpregions, which may indicate the activation of the differentiation andproliferation of B-cell populations. Immunohistochemical studies using aB cell marker, anti-CD45R(B220), are used to determine whether anyphysiological changes to splenic cells, such as splenic disorganization,are due to increased B-cell representation within loosely defined B-cellzones that infiltrate established T-cell regions.

Flow cytometric analyses of the spleens from D-SLAM protein-treated miceis used to indicate whether D-SLAM protein specifically increases theproportion of ThB+, CD45R(B220)dull B cells over that which is observedin control mice.

Likewise, a predicted consequence of increased mature B-cellrepresentation in vivo is a relative increase in serum Ig titers.Accordingly, serum IgM and IgA levels are compared between buffer andD-SLAM protein-treated mice.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 33 D-SLAM Functions as an Inhibitor of B Cell Proliferation inthe Presence of BLyS

To further examine the inhibitory activity of D-SLAM on B cells, theeffect of D-SLAM on BLyS activated B cells was examined.

Human tonsillar B cells were purified by magnetic bead (MACS) depletionof CD3-positive cells. The resulting cell population was routinelygreater than 95% B cells as assessed by expression of CD19 and CD20.Various dilutions of human recombinant D-SLAM or D-SLAM.Fc (M1-D233 ofSEQ ID NO:2 fused to the Fc portion) were placed into individual wellsof a 96-well plate to which was added 10⁵ B cells suspended in culturemedium (RPMI 1640 containing 10% FBS, 5×10⁻⁵M 2ME, 100 U/ml penicillin,100 microgram/ml streptomycin, and 10⁻⁵ dilution of Pansorbin (SAC) or10⁻⁵ dilution of Pansorbin (SAC) and BLyS (100 ng/ml) in a total volumeof 150 microliters. SAC and SAC+BLyS stimulated B cells are incubatedwith or without increasing concentrations of D-SLAM.Fc or OX40.Fc.Proliferation was quantitated by a 20 h pulse (1 microCi/well) of³H-thymidine (6.7 Ci/mM) beginning 72 h post factor addition.

Results: Addition of D-SLAM.Fc to the cell culture induced a strongdose-dependent inhibition of BLyS induced cell proliferative activity.Treatment with OX40.Fc showed no inhibitory effect.

Example 34 T Cell Proliferation Assay

A CD3-induced proliferation assay is performed on PBMCs and is measuredby the uptake of ³H-thymidine. The assay is performed as follows.Ninety-six well plates are coated with 100 μl/well of mAb to CD3 (HIT3a,Pharmingen) or isotype-matched control mAb (B33.1) overnight at 4° C. (1μg/ml in 0.05M bicarbonate buffer, pH 9.5), then washed three times withPBS. PBMC are isolated by F/H gradient centrifugation from humanperipheral blood and added to quadruplicate wells (5×10⁴/well) of mAbcoated plates in RPMI containing 10% FCS and P/S in the presence ofvarying concentrations of D-SLAM protein (total volume 200 μl). Relevantprotein buffer and medium alone are controls. After 48 hr. culture at37° C., plates are spun for 2 min. at 1000 rpm and 100 μl of supernatantis removed and stored −20° C. for measurement of IL-2 (or othercytokines) if effect on proliferation is observed. Wells aresupplemented with 100 μl of medium containing 0.5 μCi of ³H-thymidineand cultured at 37° C. for 18-24 hr. Wells are harvested andincorporation of ³H-thymidine used as a measure of proliferation.Anti-CD3 alone is the positive control for proliferation. IL-2 (100U/ml) is also used as a control which enhances proliferation. Controlantibody which does not induce proliferation of T cells is used as thenegative controls for the effects of D-SLAM proteins.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 35 Effect of D-SLAM on the Expression of MHC Class II,Costimulatory and Adhesion Molecules and Cell Differentiation ofMonocytes and Monocyte-Derived Human Dendritic Cells

Dendritic cells are generated by the expansion of proliferatingprecursors found in the peripheral blood: adherent PBMC or elutriatedmonocytic fractions are cultured for 7-10 days with GM-CSF (50 ng/ml)and IL-4 (20 ng/ml). These dendritic cells have the characteristicphenotype of immature cells (expression of CD1, CD80, CD86, CD40 and MHCclass II antigens). Treatment with activating factors, such as TNF-a,causes a rapid change in surface phenotype (increased expression of MHCclass I and II, costimulatory and adhesion molecules, downregulation ofFCγRII, upregulation of CD83). These changes correlate with increasedantigen-presenting capacity and with functional maturation of thedendritic cells.

FACS analysis of surface antigens is performed as follows. Cells aretreated 1-3 days with increasing concentrations of D-SLAM or LPS(positive control), washed with PBS containing 1% BSA and 0.02 mM sodiumazide, and then incubated with 1:20 dilution of appropriate FITC- orPE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

Effect on the production of cytokines. Cytokines generated by dendriticcells, in particular IL-12, are important in the initiation of T-celldependent immune responses. IL-12 strongly influences the development ofTh1 helper T-cell immune response, and induces cytotoxic T and NK cellfunction. An ELISA is used to measure the IL-12 release as follows.Dendritic cells (10⁶/ml) are treated with increasing concentrations ofD-SLAM for 24 hours. LPS (100 ng/ml) is added to the cell culture aspositive control. Supernatants from the cell cultures are then collectedand analyzed for IL-12 content using commercial ELISA kit (e.g, R & DSystems (Minneapolis, Minn.)). The standard protocols provided with thekits are used.

Effect on the expression of MHC Class II, costimulatory and adhesionmolecules. Three major families of cell surface antigens can beidentified on monocytes: adhesion molecules, molecules involved inantigen presentation, and Fc receptor. Modulation of the expression ofMHC class II antigens and other costimulatory molecules, such as B7 andICAM-1, may result in changes in the antigen presenting capacity ofmonocytes and ability to induce T cell activation. Increase expressionof Fc receptors may correlate with improved monocyte cytotoxic activity,cytokine release and phagocytosis.

FACS analysis is used to examine the surface antigens as follows.Monocytes are treated 1-5 days with increasing concentrations of D-SLAMor LPS (positive control), washed with PBS containing 1% BSA and 0.02 mMsodium azide, and then incubated with 1:20 dilution of appropriate FITC-or PE-labeled monoclonal antibodies for 30 minutes at 4° C. After anadditional wash, the labeled cells are analyzed by flow cytometry on aFACScan (Becton Dickinson).

Monocyte activation and/or increased survival. Assays for molecules thatactivate (or alternatively, inactivate) monocytes and/or increasemonocyte survival (or alternatively, decrease monocyte survival) areknown in the art and may routinely be applied to determine whether amolecule of the invention functions as an inhibitor or activator ofmonocytes. D-SLAM, agonists, or antagonists of D-SLAM can be screenedusing the three assays described below. For each of these assays,Peripheral blood mononuclear cells (PBMC) are purified from single donorleukopacks (American Red Cross, Baltimore, Md.) by centrifugationthrough a Histopaque gradient (Sigma). Monocytes are isolated from PBMCby counterflow centrifugal elutriation.

Monocyte Survival Assay. Human peripheral blood monocytes progressivelylose viability when cultured in absence of serum or other stimuli. Theirdeath results from internally regulated process (apoptosis). Addition tothe culture of activating factors, such as TNF-alpha dramaticallyimproves cell survival and prevents DNA fragmentation. Propidium iodide(PI) staining is used to measure apoptosis as follows. Monocytes arecultured for 48 hours in polypropylene tubes in serum-free medium(positive control), in the presence of 100 ng/ml TNF-alpha (negativecontrol), and in the presence of varying concentrations of the compoundto be tested. Cells are suspended at a concentration of 2×10⁶/ml in PBScontaining PI at a final concentration of 5 μg/ml, and then incubaed atroom temperature for 5 minutes before FACScan analysis. PI uptake hasbeen demonstrated to correlate with DNA fragmentation in thisexperimental paradigm.

Effect on cytokine release. An important function ofmonocytes/macrophages is their regulatory activity on other cellularpopulations of the immune system through the release of cytokines afterstimulation. An ELISA to measure cytokine release is performed asfollows. Human monocytes are incubated at a density of 5×10⁵ cells/mlwith increasing concentrations of D-SLAM and under the same conditions,but in the absence of D-SLAM. For IL-12 production, the cells are primedovernight with IFN (100 U/ml) in presence of D-SLAM. LPS (10 ng/ml) isthen added. Conditioned media are collected after 24 h and kept frozenuntil use. Measurement of TNF-alpha, IL-10, MCP-1 and IL-8 is thenperformed using a commercially available ELISA kit (e.g, R & D Systems(Minneapolis, Minn.)) and applying the standard protocols provided withthe kit.

Oxidative burst. Purified monocytes are plated in 96-w plate at 2-1×10⁵cell/well. Increasing concentrations of D-SLAM are added to the wells ina total volume of 0.2 ml culture medium (RPMI 1640+10% FCS, glutamineand antibiotics). After 3 days incubation, the plates are centrifugedand the medium is removed from the wells. To the macrophage monolayers,0.2 ml per well of phenol red solution (140 mM NaCl, 10 mM potassiumphosphate buffer pH 7.0, 5.5 mM dextrose, 0.56 mM phenol red and 19 U/mlof HRPO) is added, together with the stimulant (200 nM PMA). The platesare incubated at 37° C. for 2 hours and the reaction is stopped byadding 20 μl 1N NaOH per well. The absorbance is read at 610 nm. Tocalculate the amount of H₂O₂ produced by the macrophages, a standardcurve of a H₂O₂ solution of known molarity is performed for eachexperiment.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Results

As shown below, D-SLAM appears to specifically bind monocyte-deriveddendritic cells, while in this experiment, D-SLAM did not bind tomonocytes, T cells, or a variety of cell lines.

Mean Fluorescence Control D-SLAM Dendritic cells 3.94 10.95 Monocytes3.18 3.18 T cells 10.84 10.85 CD3 activ. T cells 5.94 5.93 IM-9 4.1 4.51K562 2.65 2.89 SupT13 2.63 3.76 Jurkat 3.37 3.62 THP-1 3.92 4.71

Moreover, following incubation of dendritic cells with D-SLAM, anupregulation of a number of cell surface markers that are indicative ofdendritic cell maturation or activation are observed (see below). Thus,D-SLAM may be involved in dendritic cell activation and maturation.

Mean Fluorescence Controls HLA-DR CD54 CD86 CD83 Untreated {fraction(6/5)} 1597 325 23 4 D-SLAM 10 {fraction (6/4)} 2432 488 43 5 D-SLAM 100{fraction (5/3)} 3545 309 154 10 D-SLAM 1000 {fraction (8/3)} 2952 657258 18

Additional data suggests that D-SLAM may induce TNF-alpha production bymonocytes, and that D-SLAM may bind to itself in a homotypicassociation. Therefore, D-SLAM would likely activate any cell, includingnon-hematopoietic cells, such as stromal cells, having D-SLAM expressedon the cell surface.

Thus, activating/maturing dendritic cells by increasing the amount ofD-SLAM in a patient could be useful in the treatment, diagnosis,detection, and/or prevention of cancer and modulation the immuneresponse. For example, if increasing the amount of D-SLAM elevates cellsurface expression of dendritic cell markers, such as CD83, immuneresponses against tumors may be activated. In such an instance,treatment of patients having tumors with a soluble version of D-SLAMcould lead to upregulation of CD83 on dendritic cells and result inenhanced recognition of tumors leading to an enhanced rejection/killingof the tumor.

Example 36 D-SLAM Biological Effects

Astrocyte and Neuronal Assays.

Recombinant D-SLAM, expressed and purified as described above, can betested for activity in promoting the survival, neurite outgrowth, orphenotypic differentiation of cortical neuronal cells and for inducingthe proliferation of glial fibrillary acidic protein immunopositivecells, astrocytes. The selection of cortical cells for the bioassay isbased on the prevalent expression of FGF-1 and FGF-2 in corticalstructures and on the previously reported enhancement of corticalneuronal survival resulting from FGF-2 treatment. A thymidineincorporation assay, for example, can be used to elucidate D-SLAM'sactivity on these cells.

Moreover, previous reports describing the biological effects of FGF-2(basic FGF) on cortical or hippocampal neurons in vitro havedemonstrated increases in both neuron survival and neurite outgrowth(Walicke, P. et al., “Fibroblast growth factor promotes survival ofdissociated hippocampal neurons and enhances neurite extension.” Proc.Natl. Acad. Sci. USA 83:3012-3016. (1986), assay herein incorporated byreference in its entirety). However, reports from experiments done onPC-12 cells suggest that these two responses are not necessarilysynonymous and may depend on not only which FGF is being tested but alsoon which receptor(s) are expressed on the target cells. Using theprimary cortical neuronal culture paradigm, the ability of D-SLAM toinduce neurite outgrowth can be compared to the response achieved withFGF-2 using, for example, a thymidine incorporation assay.

Fibroblast and Endothelial Cell Assays.

Human lung fibroblasts are obtained from Clonetics (San Diego, Calif.)and maintained in growth media from Clonetics. Dermal microvascularendothelial cells are obtained from Cell Applications (San Diego,Calif.). For proliferation assays, the human lung fibroblasts and dermalmicrovascular endothelial cells can be cultured at 5,000 cells/well in a96-well plate for one day in growth medium. The cells are then incubatedfor one day in 0.1% BSA basal medium. After replacing the medium withfresh 0.1% BSA medium, the cells are incubated with the test proteinsfor 3 days. Alamar Blue (Alamar Biosciences, Sacramento, Calif.) isadded to each well to a final concentration of 10%. The cells areincubated for 4 hr. Cell viability is measured by reading in a CytoFluorfluorescence reader. For the PGE₂ assays, the human lung fibroblasts arecultured at 5,000 cells/well in a 96-well plate for one day. After amedium change to 0.1% BSA basal medium, the cells are incubated withFGF-2 or D-SLAM with or without IL-1a for 24 hours. The supernatants arecollected and assayed for PGE₂ by EIA kit (Cayman, Ann Arbor, Mich.).For the IL-6 assays, the human lung fibroblasts are cultured at 5,000cells/well in a 96-well plate for one day. After a medium change to 0.1%BSA basal medium, the cells are incubated with FGF-2 or D-SLAM with orwithout IL-1a for 24 hours. The supernatants are collected and assayedfor IL-6 by ELISA kit (Endogen, Cambridge, Mass.).

Human lung fibroblasts are cultured with FGF-2 or D-SLAM for 3 days inbasal medium before the addition of Alamar Blue to assess effects ongrowth of the fibroblasts. FGF-2 should show a stimulation at 10-2500ng/ml which can be used to compare stimulation with D-SLAM.

Parkinson Models.

The loss of motor function in Parkinson's disease is attributed to adeficiency of striatal dopamine resulting from the degeneration of thenigrostriatal dopaminergic projection neurons. An animal model forParkinson's that has been extensively characterized involves thesystemic administration of 1-methyl-4 phenyl 1,2,3,6-tetrahydropyridine(MPTP). In the CNS, MPTP is taken-up by astrocytes and catabolized bymonoamine oxidase B to 1-methyl-4-phenyl pyridine (MPP⁺) and released.Subsequently, MPP⁺ is actively accumulated in dopaminergic neurons bythe high-affinity reuptake transporter for dopamine. MPP⁺ is thenconcentrated in mitochondria by the electrochemical gradient andselectively inhibits nicotidamide adenine disphosphate: ubiquinoneoxidoreductionase (complex I), thereby interfering with electrontransport and eventually generating oxygen radicals.

It has been demonstrated in tissue culture paradigms that FGF-2 (basicFGF) has trophic activity towards nigral dopaminergic neurons (Ferrariet al., Dev. Biol. 1989). Recently, Dr. Unsicker's group hasdemonstrated that administering FGF-2 in gel foam implants in thestriatum results in the near complete protection of nigral dopaminergicneurons from the toxicity associated with MPTP exposure (Otto andUnsicker, J. Neuroscience, 1990).

Based on the data with FGF-2, D-SLAM can be evaluated to determinewhether it has an action similar to that of FGF-2 in enhancingdopaminergic neuronal survival in vitro and it can also be tested invivo for protection of dopaminergic neurons in the striatum from thedamage associated with MPTP treatment. The potential effect of D-SLAM isfirst examined in vitro in a dopaminergic neuronal cell cultureparadigm. The cultures are prepared by dissecting the midbrain floorplate from gestation day 14 Wistar rat embryos. The tissue isdissociated with trypsin and seeded at a density of 200,000 cells/cm² onpolyorthinine-laminin coated glass coverslips. The cells are maintainedin Dulbecco's Modified Eagle's medium and F12 medium containing hormonalsupplements (N1). The cultures are fixed with paraformaldehyde after 8days in vitro and are processed for tyrosine hydroxylase, a specificmarker for dopminergic neurons, immunohistochemical staining.Dissociated cell cultures are prepared from embryonic rats. The culturemedium is changed every third day and the factors are also added at thattime.

Since the dopaminergic neurons are isolated from animals at gestationday 14, a developmental time which is past the stage when thedopaminergic precursor cells are proliferating, an increase in thenumber of tyrosine hydroxylase immunopositive neurons would represent anincrease in the number of dopaminergic neurons surviving in vitro.Therefore, if D-SLAM acts to prolong the survival of dopaminergicneurons, it would suggest that D-SLAM may be involved in Parkinson'sDisease.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 37 The Effect of D-SLAM on the Growth of Vascular EndothelialCells

On day 1, human umbilical vein endothelial cells (HUVEC) are seeded at2-5×10⁴ cells/35 mm dish density in M199 medium containing 4% fetalbovine serum (FBS), 16 units/ml heparin, and 50 units/ml endothelialcell growth supplements (ECGS, Biotechnique, Inc.). On day 2, the mediumis replaced with M199 containing 10% FBS, 8 units/ml heparin. D-SLAMprotein of SEQ ID NO. 2, and positive controls, such as VEGF and basicFGF (bFGF) are added, at varying concentrations. On days 4 and 6, themedium is replaced. On day 8, cell number is determined with a CoulterCounter.

An increase in the number of HUVEC cells indicates that D-SLAM mayproliferate vascular endothelial cells.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 38 Stimulatory Effect of D-SLAM on the Proliferation of VascularEndothelial Cells

For evaluation of mitogenic activity of growth factors, the colorimetricMTS(3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)assay with the electron coupling reagent PMS (phenazine methosulfate)was performed (CellTiter 96 AQ, Promega). Cells are seeded in a 96-wellplate (5,000 cells/well) in 0.1 mL serum-supplemented medium and areallowed to attach overnight. After serum-starvation for 12 hours in 0.5%FBS, conditions (bFGF, VEGF₁₆₅ or D-SLAM in 0.5% FBS) with or withoutHeparin (8 U/ml) are added to wells for 48 hours. 20 mg of MTS/PMSmixture (1:0.05) are added per well and allowed to incubate for 1 hourat 37° C. before measuring the absorbance at 490 nm in an ELISA platereader. Background absorbance from control wells (some media, no cells)is subtracted, and seven wells are performed in parallel for eachcondition. See, Leak et al. In Vitro Cell. Dev. Biol. 30A:512-518(1994).

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 39 Inhibition of PDGF-Induced Vascular Smooth Muscle CellProliferation Stimulatory Effect

HAoSMC proliferation can be measured, for example, by BrdUrdincorporation. Briefly, subconfluent, quiescent cells grown on the4-chamber slides are transfected with CRP or FITC-labeled AT2-3LP. Then,the cells are pulsed with 10% calf serum and 6 mg/ml BrdUrd. After 24 h,immunocytochemistry is performed by using BrdUrd Staining Kit (ZymedLaboratories). In brief, the cells are incubated with the biotinylatedmouse anti-BrdUrd antibody at 4° C. for 2 h after being exposed todenaturing solution and then incubated with the streptavidin-peroxidaseand diaminobenzidine. After counterstaining with hematoxylin, the cellsare mounted for microscopic examination, and the BrdUrd-positive cellsare counted. The BrdUrd index is calculated as a percent of theBrdUrd-positive cells to the total cell number. In addition, thesimultaneous detection of the BrdUrd staining (nucleus) and the FITCuptake (cytoplasm) is performed for individual cells by the concomitantuse of bright field illumination and dark field-UV fluorescentillumination. See, Hayashida et al., J. Biol. Chem.6:271(36):21985-21992 (1996).

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 40 Stimulation of Endothelial Migration

This example will be used to explore the possibility that D-SLAM maystimulate lymphatic endothelial cell migration.

Endothelial cell migration assays are performed using a 48 wellmicrochemotaxis chamber (Neuroprobe Inc., Cabin John, MD; Falk, W., etal., J. Immunological Methods 1980;33:239-247).Polyvinylpyrrolidone-free polycarbonate filters with a pore size of 8 um(Nucleopore Corp. Cambridge, Mass.) are coated with 0.1% gelatin for atleast 6 hours at room temperature and dried under sterile air. Testsubstances are diluted to appropriate concentrations in M199supplemented with 0.25% bovine serum albumin (BSA), and 25 ul of thefinal dilution is placed in the lower chamber of the modified Boydenapparatus. Subconfluent, early passage (2-6) HUVEC or BMEC cultures arewashed and trypsinized for the minimum time required to achieve celldetachment. After placing the filter between lower and upper chamber,2.5×10⁵ cells suspended in 50 ul M199 containing 1% FBS are seeded inthe upper compartment. The apparatus is then incubated for 5 hours at37° C. in a humidified chamber with 5% CO₂ to allow cell migration.After the incubation period, the filter is removed and the upper side ofthe filter with the non-migrated cells is scraped with a rubberpoliceman. The filters are fixed with methanol and stained with a Giemsasolution (Diff-Quick, Baxter, McGraw Park, Ill.). Migration isquantified by counting cells of three random high-power fields (40×) ineach well, and all groups are performed in quadruplicate.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 41 Stimulation of Nitric Oxide Production by Endothelial Cells

Nitric oxide released by the vascular endothelium is believed to be amediator of vascular endothelium relaxation. Thus, D-SLAM activity canbe assayed by determining nitric oxide production by endothelial cellsin response to D-SLAM.

Nitric oxide is measured in 96-well plates of confluent microvascularendothelial cells after 24 hours starvation and a subsequent 4 hrexposure to various levels of a positive control (such as VEGF-1) andD-SLAM. Nitric oxide in the medium is determined by use of the Griessreagent to measure total nitrite after reduction of nitric oxide-derivednitrate by nitrate reductase. The effect of D-SLAM on nitric oxiderelease is examined on HUVEC.

Briefly, NO release from cultured HUVEC monolayer is measured with aNO-specific polarographic electrode connected to a NO meter (Iso-NO,World Precision Instruments Inc.) (1049). Calibration of the NO elementsis performed according to the following equation: 2 KNO2+2 KI+2H₂SO₄ 6 2NO+12+2H₂O+2 K₂SO₄

The standard calibration curve is obtained by adding gradedconcentrations of KNO₂ (0, 5, 10, 25, 50, 100, 250, and 500 nmol/L) intothe calibration solution containing K₁ and H₂SO₄. The specificity of theIso-NO electrode to NO is previously determined by measurement of NOfrom authentic NO gas (1050). The culture medium is removed and KUVECsare washed twice with Dulbecco's phosphate buffered saline. The cellsare then bathed in 5 ml of filtered Krebs-Henseleit solution in 6-wellplates, and the cell plates are kept on a slide warmer (Lab LineInstruments Inc.) To maintain the temperature at 37° C. The NO sensorprobe is inserted vertically into the wells, keeping the tip of theelectrode 2 mm under the surface of the solution, before addition of thedifferent conditions. S-nitroso acetyl penicillamin (SNAP) is used as apositive control. The amount of released NO is expressed as picomolesper 1×10⁶ endothelial cells. All values reported are means of four tosix measurements in each group (number of cell culture wells). See, Leaket al. Biochem. and Biophys. Res. Comm. 217:96-105 (1995).

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 42 Effect of D-SLAM on Cord Formation in Angiogenesis

Another step in angiogenesis is cord formation, marked bydifferentiation of endothelial cells. This bioassay measures the abilityof microvascular endothelial cells to form capillary-like structures(hollow structures) when cultured in vitro.

CADMEC (microvascular endothelial cells) are purchased from CellApplications, Inc. as proliferating (passage 2) cells and are culturedin Cell Applications' CADMEC Growth Medium and used at passage 5. Forthe in vitro angiogenesis assay, the wells of a 48-well cell cultureplate are coated with Cell Applications' Attachment Factor Medium (200ml/well) for 30 min. at 37° C. CADMEC are seeded onto the coated wellsat 7,500 cells/well and cultured overnight in Growth Medium. The GrowthMedium is then replaced with 300 mg Cell Applications' Chord FormationMedium containing control buffer or D-SLAM (0.1 to 100 ng/ml) and thecells are cultured for an additional 48 hr. The numbers and lengths ofthe capillary-like chords are quantitated through use of the BoeckelerVIA-170 video image analyzer. All assays are done in triplicate.

Commercial (R&D) VEGF (50 ng/ml) is used as a positive control.b-esteradiol (1 ng/ml) is used as a negative control. The appropriatebuffer (without protein) is also utilized as a control.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 43 Angiogenic Effect on Chick Chorioallantoic Membrane

Chick chorioallantoic membrane (CAM) is a well-established system toexamine angiogenesis. Blood vessel formation on CAM is easily visibleand quantifiable. The ability of D-SLAM to stimulate angiogenesis in CAMcan be examined.

Fertilized eggs of the White Leghorn chick (Gallus gallus) and theJapanese qual (Coturnix coturnix) are incubated at 37.8° C. and 80%humidity. Differentiated CAM of 16-day-old chick and 13-day-old qualembryos is studied with the following methods.

On Day 4 of development, a window is made into the egg shell of chickeggs. The embryos are checked for normal development and the eggs sealedwith cellotape. They are further incubated until Day 13. Thermanoxcoverslips (Nunc, Naperville, Ill.) are cut into disks of about 5 mm indiameter. Sterile and salt-free growth factors are dissolved indistilled water and about 3.3 mg/5 ml are pipetted on the disks. Afterair-drying, the inverted disks are applied on CAM. After 3 days, thespecimens are fixed in 3% glutaraldehyde and 2% formaldehyde and rinsedin 0.12 M sodium cacodylate buffer. They are photographed with a stereomicroscope [Wild M8] and embedded for semi- and ultrathin sectioning asdescribed above. Controls are performed with carrier disks alone.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 44 Angiogenesis Assay Using a Matrigel Implant in Mouse

In vivo angiogenesis assay of D-SLAM measures the ability of an existingcapillary network to form new vessels in an implanted capsule of murineextracellular matrix material (Matrigel). The protein is mixed with theliquid Matrigel at 4° C. and the mixture is then injected subcutaneouslyin mice where it solidifies. After 7 days, the solid “plug” of Matrigelis removed and examined for the presence of new blood vessels. Matrigelis purchased from Becton Dickinson Labware/Collaborative BiomedicalProducts.

When thawed at 4° C. the Matrigel material is a liquid. The Matrigel ismixed with D-SLAM at 150 ng/ml at 4° C. and drawn into cold 3 mlsyringes. Female C57Bi/6 mice approximately 8 weeks old are injectedwith the mixture of Matrigel and experimental protein at 2 sites at themidventral aspect of the abdomen (0.5 ml/site). After 7 days, the miceare sacrificed by cervical dislocation, the Matrigel plugs are removedand cleaned (i.e., all clinging membranes and fibrous tissue isremoved). Replicate whole plugs are fixed in neutral buffered 10%formaldehyde, embedded in paraffin and used to produce sections forhistological examination after staining with Masson's Trichrome. Crosssections from 3 different regions of each plug are processed. Selectedsections are stained for the presence of vWF. The positive control forthis assay is bovine basic FGF (150 ng/ml). Matrigel alone is used todetermine basal levels of angiogenesis.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 45 Rescue ofischemia in Rabbit Lower Limb Model

To study the in vivo effects of D-SLAM on ischemia, a rabbit hindlimbischemia model is created by surgical removal of one femoral arteries asdescribed previously (Takeshita, S. et al., Am J. Pathol 147:1649-1660(1995)). The excision of the femoral artery results in retrogradepropagation of thrombus and occlusion of the external iliac artery.Consequently, blood flow to the ischemic limb is dependent uponcollateral vessels originating from the internal iliac artery(Takeshita, S. et al. Am J. Pathol 147:1649-1660 (1995)). An interval of10 days is allowed for post-operative recovery of rabbits anddevelopment of endogenous collateral vessels. At 10 day post-operatively(day 0), after performing a baseline angiogram, the internal iliacartery of the ischemic limb is transfected with 500 mg naked D-SLAMexpression plasmid by arterial gene transfer technology using ahydrogel-coated balloon catheter as described (Riessen, R. et al. HumGene Ther. 4:749-758 (1993); Leclerc, G. et al. J. Clin. Invest. 90:936-944 (1992)). When D-SLAM is used in the treatment, a single bolus of500 mg D-SLAM protein or control is delivered into the internal iliacartery of the ischemic limb over a period of 1 min. through an infusioncatheter. On day 30, various parameters are measured in these rabbits:(a) BP ratio—The blood pressure ratio of systolic pressure of theischemic limb to that of normal limb; (b) Blood Flow and FlowReserve—Resting FL: the blood flow during undilated condition and MaxFL: the blood flow during fully dilated condition (also an indirectmeasure of the blood vessel amount) and Flow Reserve is reflected by theratio of max FL: resting FL; (c) Angiographic Score—This is measured bythe angiogram of collateral vessels. A score is determined by thepercentage of circles in an overlaying grid that with crossing opacifiedarteries divided by the total number m the rabbit thigh; (d) Capillarydensity—The number of collateral capillaries determined in lightmicroscopic sections taken from hindlimbs.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 46 Effect of D-SLAM on Vasodilation

Since dilation of vascular endothelium is important in reducing bloodpressure, the ability of D-SLAM to affect the blood pressure inspontaneously hypertensive rats (SHR) is examined. Increasing doses (0,10, 30, 100, 300, and 900 mg/kg) of the D-SLAM are administered to 13-14week old spontaneously hypertensive rats (SHR). Data are expressed asthe mean +/−SEM. Statistical analysis are performed with a paired t-testand statistical significance is defined as p<0.05 vs. the response tobuffer alone.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 47 Rat Ischemic Skin Flap Model

The evaluation parameters include skin blood flow, skin temperature, andfactor VIII immunohistochemistry or endothelial alkaline phosphatasereaction. D-SLAM expression, during the skin ischemia, is studied usingin situ hybridization.

The study in this model is divided into three parts as follows:

-   -   (a) Ischemic skin    -   (b) Ischemic skin wounds    -   (c) Normal wounds

The experimental protocol includes:

-   -   (a) Raising a 3×4 cm, single pedicle full-thickness random skin        flap (myocutaneous flap over the lower back of the animal).    -   (b) An excisional wounding (4-6 mm in diameter) in the ischemic        skin (skin-flap).    -   (c) Topical treatment with D-SLAM of the excisional wounds (day        0, 1, 2, 3, 4 post-wounding) at the following various dosage        ranges: 1 mg to 100 mg.    -   (d) Harvesting the wound tissues at day 3, 5, 7, 10, 14 and 21        post-wounding for histological, immunohistochemical, and in situ        studies.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 48 Peripheral Arterial Disease Model

Angiogenic therapy using D-SLAM is a novel therapeutic strategy toobtain restoration of blood flow around the ischemia in case ofperipheral arterial diseases. The experimental protocol includes:

-   -   (a) One side of the femoral artery is ligated to create ischemic        muscle of the hindlimb, the other side of hindlimb serves as a        control.    -   (b) D-SLAM protein, in a dosage range of 20 mg-500 mg, is        delivered intravenously and/or intramuscularly 3 times (perhaps        more) per week for 2-3 weeks.    -   (c) The ischemic muscle tissue is collected after ligation of        the femoral artery at 1, 2, and 3 weeks for the analysis of        D-SLAM expression and histology. Biopsy is also performed on the        other side of normal muscle of the contralateral hindlimb.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 49 Ischemic Myocardial Disease Model

D-SLAM is evaluated as a potent mitogen capable of stimulating thedevelopment of collateral vessels, and restructuring new vessels aftercoronary artery occlusion. Alteration of D-SLAM expression isinvestigated in situ. The experimental protocol includes:

The heart is exposed through a left-side thoracotomy in the rat.Immediately, the left coronary artery is occluded with a thin suture(6-0) and the thorax is closed.

D-SLAM protein, in a dosage range of 20 mg-500 mg, is deliveredintravenously and/or intramuscularly 3 times (perhaps more) per week for2-4 weeks.

Thirty days after the surgery, the heart is removed and cross-sectionedfor morphometric and in situ analyzes.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 50 Rat Corneal Wound Healing Model

This animal model shows the effect of D-SLAM on neovascularization. Theexperimental protocol includes:

-   -   (a) Making a 1-1.5 mm long incision from the center of cornea        into the stromal layer.    -   (b) Inserting a spatula below the lip of the incision facing the        outer corner of the eye.    -   (c) Making a pocket (its base is 1-1.5 mm form the edge of the        eye).    -   (d) Positioning a pellet, containing 50 ng-5 ug of D-SLAM,        within the pocket.

D-SLAM treatment can also be applied topically to the corneal wounds ina dosage range of 20 mg-500 mg (daily treatment for five days).

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 51 Diabetic Mouse and Glucocorticoid-Impaired Wound HealingModels Diabetic db+/db+ Mouse Model

To demonstrate that D-SLAM accelerates the healing process, thegenetically diabetic mouse model of wound healing is used. The fullthickness wound healing model in the db+/db+ mouse is a wellcharacterized, clinically relevant and reproducible model of impairedwound healing. Healing of the diabetic wound is dependent on formationof granulation tissue and re-epithelialization rather than contraction(Gartner, M. H. et al., J. Surg. Res. 52:389 (1992); Greenhalgh, D. G.et al., Am. J. Pathol. 136:1235 (1990)).

The diabetic animals have many of the characteristic features observedin Type II diabetes mellitus. Homozygous (db+/db+) mice are obese incomparison to their normal heterozygous (db+/+m) littermates. Mutantdiabetic (db+/db+) mice have a single autosomal recessive mutation onchromosome 4 (db+) (Coleman et al. Proc. Natl. Acad. Sci. USA 77:283-293(1982)). Animals show polyphagia, polydipsia and polyuria. Mutantdiabetic mice (db+/db+) have elevated blood glucose, increased or normalinsulin levels, and suppressed cell-mediated immunity (Mandel et al., J.Immunol. 120:1375 (1978); Debray-Sachs, M. et al., Clin. Exp. Immunol.51(1):1-7 (1983); Leiter et al., Am. J. of Pathol. 114:46-55 (1985)).Peripheral neuropathy, myocardial complications, and microvascularlesions, basement membrane thickening and glomerular filtrationabnormalities have been described in these animals (Norido, F. et al.,Exp. Neurol. 83(2):221-232 (1984); Robertson et al., Diabetes29(1):60-67 (1980); Giacomelli et al., Lab Invest. 40(4):460-473 (1979);Coleman, D. L., Diabetes 31 (Suppl):1-6 (1982)). These homozygousdiabetic mice develop hyperglycemia that is resistant to insulinanalogous to human type II diabetes (Mandel et al., J. Immunol.120:1375-1377 (1978)).

The characteristics observed in these animals suggests that healing inthis model may be similar to the healing observed in human diabetes(Greenhalgh, et al., Am. J. of Pathol. 136:1235-1246 (1990)).

Genetically diabetic female C57BL/KsJ (db+/db+) mice and theirnon-diabetic (db+/+m) heterozygous littermates are used in this study(Jackson Laboratories). The animals are purchased at 6 weeks of age andare 8 weeks old at the beginning of the study. Animals are individuallyhoused and received food and water ad libitum. All manipulations areperformed using aseptic techniques. The experiments are conductedaccording to the rules and guidelines of Human Genome Sciences, Inc.Institutional Animal Care and Use Committee and the Guidelines for theCare and Use of Laboratory Animals.

Wounding protocol is performed according to previously reported methods(Tsuboi, R. and Rifkin, D. B., J. Exp. Med. 172:245-251 (1990)).Briefly, on the day of wounding, animals are anesthetized with anintraperitoneal injection of Avertin (0.01 mg/mL), 2,2,2-tribromoethanoland 2-methyl-2-butanol dissolved in deionized water. The dorsal regionof the animal is shaved and the skin washed with 70% ethanol solutionand iodine. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is then created using a Keyestissue punch. Immediately following wounding, the surrounding skin isgently stretched to eliminate wound expansion. The wounds are left openfor the duration of the experiment. Application of the treatment isgiven topically for 5 consecutive days commencing on the day ofwounding. Prior to treatment, wounds are gently cleansed with sterilesaline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of surgery and at two day intervals thereafter. Wound closure isdetermined by daily measurement on days 1-5 and on day 8. Wounds aremeasured horizontally and vertically using a calibrated Jameson caliper.Wounds are considered healed if granulation tissue is no longer visibleand the wound is covered by a continuous epithelium.

D-SLAM is administered using at a range different doses of D-SLAM, from4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology and immunohistochemistry. Tissue specimensare placed in 10% neutral buffered formalin in tissue cassettes betweenbiopsy sponges for further processing.

Three groups of 10 animals each (5 diabetic and 5 non-diabetic controls)are evaluated: 1) Vehicle placebo control, 2) D-SLAM.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total square area of the wound.Contraction is then estimated by establishing the differences betweenthe initial wound area (day 0) and that of post treatment (day 8). Thewound area on day 1 is 64 mm², the corresponding size of the dermalpunch. Calculations are made using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing a Reichert-Jung microtome. Routine hematoxylin-eosin (H&E)staining is performed on cross-sections of bisected wounds. Histologicexamination of the wounds are used to assess whether the healing processand the morphologic appearance of the repaired skin is altered bytreatment with D-SLAM. This assessment included verification of thepresence of cell accumulation, inflammatory cells, capillaries,fibroblasts, re-epithelialization and epidermal maturity (Greenhalgh, D.G. et al., Am. J. Pathol. 136:1235 (1990)). A calibrated lens micrometeris used by a blinded observer.

Tissue sections are also stained immunohistochemically with a polyclonalrabbit anti-human keratin antibody using ABC Elite detection system.Human skin is used as a positive tissue control while non-immune IgG isused as a negative control. Keratinocyte growth is determined byevaluating the extent of reepithelialization of the wound using acalibrated lens micrometer.

Proliferating cell nuclear antigen/cyclin (PCNA) in skin specimens isdemonstrated by using anti-PCNA antibody (1:50) with an ABC Elitedetection system. Human colon cancer served as a positive tissue controland human brain tissue is used as a negative tissue control. Eachspecimen included a section with omission of the primary antibody andsubstitution with non-immune mouse IgG. Ranking of these sections isbased on the extent of proliferation on a scale of 0-8, the lower sideof the scale reflecting slight proliferation to the higher sidereflecting intense proliferation.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

Steroid Impaired Rat Model

The inhibition of wound healing by steroids has been well documented invarious in vitro and in vivo systems (Wahl, S. M. Glucocorticoids andWound healing. In: Anti-Inflammatory Steroid Action: Basic and ClinicalAspects. 280-302 (1989); Wahl, S. M. et al., J. Immunol. 115: 476-481(1975); Werb, Z. et al., J. Exp. Med. 147:1684-1694 (1978)).Glucocorticoids retard wound healing by inhibiting angiogenesis,decreasing vascular permeability (Ebert, R. H., et al., An. Intern. Med.37:701-705 (1952)), fibroblast proliferation, and collagen synthesis(Beck, L. S. et al., Growth Factors. 5: 295-304 (1991); Haynes, B. F. etal., J. Clin. Invest. 61: 703-797 (1978)) and producing a transientreduction of circulating monocytes (Haynes, B. F., et al., J. Clin.Invest. 61: 703-797 (1978); Wahl, S. M., “Glucocorticoids and woundhealing”, In: Antiinflammatory Steroid Action: Basic and ClinicalAspects, Academic Press, New York, pp. 280-302 (1989)). The systemicadministration of steroids to impaired wound healing is a well establishphenomenon in rats (Beck, L. S. et al., Growth Factors. 5: 295-304(1991); Haynes, B. F., et al., J. Clin. Invest. 61: 703-797 (1978);Wahl, S. M., “Glucocorticoids and wound healing”, In: AntiinflammatorySteroid Action: Basic and Clinical Aspects, Academic Press, New York,pp. 280-302 (1989); Pierce, G. F. et al., Proc. Natl. Acad. Sci. USA 86:2229-2233 (1989)).

To demonstrate that D-SLAM can accelerate the healing process, theeffects of multiple topical applications of D-SLAM on full thicknessexcisional skin wounds in rats in which healing has been impaired by thesystemic administration of methylprednisolone is assessed.

Young adult male Sprague Dawley rats weighing 250-300 g (Charles RiverLaboratories) are used in this example. The animals are purchased at 8weeks of age and are 9 weeks old at the beginning of the study. Thehealing response of rats is impaired by the systemic administration ofmethylprednisolone (17 mg/kg/rat intramuscularly) at the time ofwounding. Animals are individually housed and received food and water adlibitum. All manipulations are performed using aseptic techniques. Thisstudy is conducted according to the rules and guidelines of Human GenomeSciences, Inc. Institutional Animal Care and Use Committee and theGuidelines for the Care and Use of Laboratory Animals.

The wounding protocol is followed according to section A, above. On theday of wounding, animals are anesthetized with an intramuscularinjection of ketamine (50 mg/kg) and xylazine (5 mg/kg). The dorsalregion of the animal is shaved and the skin washed with 70% ethanol andiodine solutions. The surgical area is dried with sterile gauze prior towounding. An 8 mm full-thickness wound is created using a Keyes tissuepunch. The wounds are left open for the duration of the experiment.Applications of the testing materials are given topically once a day for7 consecutive days commencing on the day of wounding and subsequent tomethylprednisolone administration. Prior to treatment, wounds are gentlycleansed with sterile saline and gauze sponges.

Wounds are visually examined and photographed at a fixed distance at theday of wounding and at the end of treatment. Wound closure is determinedby daily measurement on days 1-5 and on day 8. Wounds are measuredhorizontally and vertically using a calibrated Jameson caliper. Woundsare considered healed if granulation tissue is no longer visible and thewound is covered by a continuous epithelium.

D-SLAM is administered using at a range different doses of D-SLAM, from4 mg to 500 mg per wound per day for 8 days in vehicle. Vehicle controlgroups received 50 mL of vehicle solution.

Animals are euthanized on day 8 with an intraperitoneal injection ofsodium pentobarbital (300 mg/kg). The wounds and surrounding skin arethen harvested for histology. Tissue specimens are placed in 10% neutralbuffered formalin in tissue cassettes between biopsy sponges for furtherprocessing.

Four groups of 10 animals each (5 with methylprednisolone and 5 withoutglucocorticoid) are evaluated: 1) Untreated group 2) Vehicle placebocontrol 3) D-SLAM treated groups.

Wound closure is analyzed by measuring the area in the vertical andhorizontal axis and obtaining the total area of the wound. Closure isthen estimated by establishing the differences between the initial woundarea (day 0) and that of post treatment (day 8). The wound area on day 1is 64 mm², the corresponding size of the dermal punch. Calculations aremade using the following formula:[Open area on day 8]−[Open area on day 1]/[Open area on day 1]

Specimens are fixed in 10% buffered formalin and paraffin embeddedblocks are sectioned perpendicular to the wound surface (5 mm) and cutusing an Olympus microtome. Routine hematoxylin-eosin (H&E) staining isperformed on cross-sections of bisected wounds. Histologic examinationof the wounds allows assessment of whether the healing process and themorphologic appearance of the repaired skin is improved by treatmentwith D-SLAM. A calibrated lens micrometer is used by a blinded observerto determine the distance of the wound gap.

Experimental data are analyzed using an unpaired t test. A p value of<0.05 is considered significant.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 52 Lymphadema Animal Model

The purpose of this experimental approach is to create an appropriateand consistent lymphedema model for testing the therapeutic effects ofD-SLAM in lymphangiogenesis and re-establishment of the lymphaticcirculatory system in the rat hind limb. Effectiveness is measured byswelling volume of the affected limb, quantification of the amount oflymphatic vasculature, total blood plasma protein, and histopathology.Acute lymphedema is observed for 7-10 days. Perhaps more importantly,the chronic progress of the edema is followed for up to 3-4 weeks.

Prior to beginning surgery, blood sample is drawn for proteinconcentration analysis. Male rats weighing approximately ˜350 g aredosed with Pentobarbital. Subsequently, the right legs are shaved fromknee to hip. The shaved area is swabbed with gauze soaked in 70% EtOH.Blood is drawn for serum total protein testing. Circumference andvolumetric measurements are made prior to injecting dye into paws aftermarking 2 measurement levels (0.5 cm above heel, at mid-pt of dorsalpaw). The intradermal dorsum of both right and left paws are injectedwith 0.05 ml of 1% Evan's Blue. Circumference and volumetricmeasurements are then made following injection of dye into paws.

Using the knee joint as a landmark, a mid-leg inguinal incision is madecircumferentially allowing the femoral vessels to be located. Forcepsand hemostats are used to dissect and separate the skin flaps. Afterlocating the femoral vessels, the lymphatic vessel that runs along sideand underneath the vessel(s) is located. The main lymphatic vessels inthis area are then electrically coagulated or suture ligated.

Using a microscope, muscles in back of the leg (near the semitendinosisand adductors) are bluntly dissected. The popliteal lymph node is thenlocated. The 2 proximal and 2 distal lymphatic vessels and distal bloodsupply of the popliteal node are then and ligated by suturing. Thepopliteal lymph node, and any accompanying adipose tissue, is thenremoved by cutting connective tissues.

Care is taken to control any mild bleeding resulting from thisprocedure. After lymphatics are occluded, the skin flaps are sealed byusing liquid skin (Vetbond) (AJ Buck). The separated skin edges aresealed to the underlying muscle tissue while leaving a gap of ˜0.5 cmaround the leg. Skin also may be anchored by suturing to underlyingmuscle when necessary.

To avoid infection, animals are housed individually with mesh (nobedding). Recovering animals are checked daily through the optimaledematous peak, which typically occurred by day 5-7. The plateauedematous peak are then observed. To evaluate the intensity of thelymphedema, the circumference and volumes of 2 designated places on eachpaw before operation and daily for 7 days are measured. The effectplasma proteins on lymphedema is determined and whether protein analysisis a useful testing perimeter is also investigated. The weights of bothcontrol and edematous limbs are evaluated at 2 places. Analysis isperformed in a blind manner.

Circumference Measurements: Under brief gas anesthetic to prevent limbmovement, a cloth tape is used to measure limb circumference.Measurements are done at the ankle bone and dorsal paw by 2 differentpeople then those 2 readings are averaged. Readings are taken from bothcontrol and edematous limbs.

Volumetric Measurements: On the day of surgery, animals are anesthetizedwith Pentobarbital and are tested prior to surgery. For dailyvolumetrics animals are under brief halothane anesthetic (rapidimmobilization and quick recovery), both legs are shaved and equallymarked using waterproof marker on legs. Legs are first dipped in water,then dipped into instrument to each marked level then measured by Buxcoedema software (Chen/Victor). Data is recorded by one person, while theother is dipping the limb to marked area.

Blood-plasma protein measurements: Blood is drawn, spun, and serumseparated prior to surgery and then at conclusion for total protein andCa2+ comparison.

Limb Weight Comparison: After drawing blood, the animal is prepared fortissue collection. The limbs are amputated using a quillitine, then bothexperimental and control legs are cut at the ligature and weighed. Asecond weighing is done as the tibio-cacaneal joint is disarticulatedand the foot is weighed.

Histological Preparations: The transverse muscle located behind the knee(popliteal) area is dissected and arranged in a metal mold, filled withfreezeGel, dipped into cold methylbutane, placed into labeled samplebags at −80EC until sectioning. Upon sectioning, the muscle is observedunder fluorescent microscopy for lymphatics.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 53 Suppression of TNF Alpha-Induced Adhesion Molecule Expressionby D-SLAM

The recruitment of lymphocytes to areas of inflammation and angiogenesisinvolves specific receptor-ligand interactions between cell surfaceadhesion molecules (CAMs) on lymphocytes and the vascular endothelium.The adhesion process, in both normal and pathological settings, followsa multi-step cascade that involves intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and endothelialleukocyte adhesion molecule-1 (E-selectin) expression on endothelialcells (EC). The expression of these molecules and others on the vascularendothelium determines the efficiency with which leukocytes may adhereto the local vasculature and extravasate into the local tissue duringthe development of an inflammatory response. The local concentration ofcytokines and growth factor participate in the modulation of theexpression of these CAMs.

Tumor necrosis factor alpha (TNF-a), a potent proinflammatory cytokine,is a stimulator of all three CAMs on endothelial cells and may beinvolved in a wide variety of inflammatory responses, often resulting ina pathological outcome.

The potential of D-SLAM to mediate a suppression of TNF-a induced CAMexpression can be examined. A modified ELISA assay which uses ECs as asolid phase absorbent is employed to measure the amount of CAMexpression on TNF-a treated ECs when co-stimulated with a member of theFGF family of proteins.

To perform the experiment, human umbilical vein endothelial cell (HUVEC)cultures are obtained from pooled cord harvests and maintained in growthmedium (EGM-2; Clonetics, San Diego, Calif.) supplemented with 10% FCSand 1% penicillin/streptomycin in a 37° C. humidified incubatorcontaining 5% CO₂. HUVECs are seeded in 96-well plates at concentrationsof 1×10⁴ cells/well in EGM medium at 37° C. for 18-24 hrs or untilconfluent. The monolayers are subsequently washed 3 times with aserum-free solution of RPMI-1640 supplemented with 100 U/ml penicillinand 100 mg/ml streptomycin, and treated with a given cytokine and/orgrowth factor(s) for 24 h at 37° C. Following incubation, the cells arethen evaluated for CAM expression.

Human Umbilical Vein Endothelial cells (HUVECs) are grown in a standard96 well plate to confluence. Growth medium is removed from the cells andreplaced with 90 ul of 199 Medium (10% FBS). Samples for testing andpositive or negative controls are added to the plate in triplicate (in10 ul volumes). Plates are incubated at 37° C. for either 5 h (selectinand integrin expression) or 24 h (integrin expression only). Plates areaspirated to remove medium and 100 μl of 0.1% paraformaldehyde-PBS (withCa++ and Mg++) is added to each well. Plates are held at 4° C. for 30min.

Fixative is then removed from the wells and wells are washed 1× withPBS(+Ca,Mg)+0.5% BSA and drained. Do not allow the wells to dry. Add 10μl of diluted primary antibody to the test and control wells.Anti-ICAM-1-Biotin, Anti-VCAM-1-Biotin and Anti-E-selectin-Biotin areused at a concentration of 10 μg/ml (1:10 dilution of 0.1 mg/ml stockantibody). Cells are incubated at 37° C. for 30 min. in a humidifiedenvironment. Wells are washed ×3 with PBS(+Ca,Mg)+0.5% BSA.

Then add 20 μl of diluted ExtrAvidin-Alkaline Phosphotase (1:5,000dilution) to each well and incubated at 37° C. for 30 min. Wells arewashed ×3 with PBS(+Ca,Mg)+0.5% BSA. 1 tablet of p-Nitrophenol PhosphatepNPP is dissolved in 5 ml of glycine buffer (pH 10.4). 100 μl of pNPPsubstrate in glycine buffer is added to each test well. Standard wellsin triplicate are prepared from the working dilution of theExtrAvidin-Alkaline Phosphotase in glycine buffer: 1:5,000(10⁰)>10^(−0.5)>10⁻¹>10^(−1.5). 5 μl of each dilution is added totriplicate wells and the resulting AP content in each well is 5.50 ng,1.74 ng, 0.55 ng, 0.18 ng. 100 μl of pNNP reagent must then be added toeach of the standard wells. The plate must be incubated at 37° C. for 4h. A volume of 50 μl of 3M NaOH is added to all wells. The results arequantified on a plate reader at 405 nm. The background subtractionoption is used on blank wells filled with glycine buffer only. Thetemplate is set up to indicate the concentration of AP-conjugate in eachstandard well [5.50 ng; 1.74 ng; 0.55 ng; 0.18 ng]. Results areindicated as amount of bound AP-conjugate in each sample.

The studies described in this example tested activity in D-SLAM protein.However, one skilled in the art could easily modify the exemplifiedstudies to test the activity of D-SLAM polynucleotides (e.g., genetherapy), agonists, and/or antagonists of D-SLAM.

Example 54 Effect of D-SLAM and its Antagonists and Agonists in TreatingGraft-Versus-Host Disease Associated Lymphoid Atrophy and Hypoplasia inMice

An analysis of the use of antagonists of D-SLAM (e.g., anti-D-SLAMantibody) to treat, prevent, detect, and/or diagnose graft-versus-hostdisease (GVHD)-associated lymphoid hypoplasia/atrophy is performedthrough the use of a C57BL/6 parent into (BALB/c X C57BL/6) F1 (CBF1)mouse model. This parent into F1 mouse model is a well-characterized andreproducible animal model of GVHD in bone marrow transplant patients,which is well know to one of ordinary skill in the art (see,Gleichemann, et al., Immunol. Today 5:324, 1984). A soluble D-SLAMantagonist is expected to induce the proliferation and differentiationof B lymphocytes, and correct the lymphoid hypoplasia and atrophyobserved in this animal model of GVHD (Piguet, et al., J. Exp. Med.166:1280 (1987); Hattori, et al., Blood 90:542 (1997)).

Initiation of the GVHD condition is induced by the intravenous injectionof approximately 1-5×10⁸ spleen cells from C57BL/6 mice into(BALB/c×C57BL/6) F1 mice (both are available from Jackson Lab, BarHarbor, Me.). Groups of 6 to 8 mice receive daily either 0.1 to 5.0mg/kg of D-SLAM antagonist or buffer control intraperitoneally,intramascullarly or intradermally starting from the days when lymphoidhypoplasia and atrophy are mild (˜day 5), moderate (˜day 12) or severe(˜day 20) following the parental cell injection. The effect of D-SLAMantagonist on lymphoid hypoplasia and atrophy of spleen is analyzed byFACS and histopathology at multiple time points (3-4) between day 10-30.Briefly, splenocytes are prepared from normal CBF1, GVHD or D-SLAMantagonist-treated mice, and stained with fluoresceinphycoerythrin-conjugated anti-H-2 Kb, biotin-conjugated anti-H-2 Kd, andFITC-conjugated anti-CD4, anti-CD8, or anti-B220, followed by aCyChrome-conjugated avidin. All of these conjugated antibodies can bepurchased from PharMingen (San Diego, Calif.). Cells are then analysison a FACScan (Becton Dickinson, San Jose, Calif.). Recipient and donorlymphocytes are identified as H-2 Kb+ Kd+ and H-2 Kb+ Kd− cells,respectively. Cell numbers of CD4+T, CD8+ T and B220+ B cells ofrecipient or donor origin are calculated from the total numbers ofsplenocytes recovered and the percentages of each subpopulation aredetermined by the three color analysis. Histological evaluation of therelative degree of tissue damage in other GVHD-associated organs (liver,skin and intestine) may be conducted after sacrificing the animals.

Finally, D-SLAM antagonist and buffer-treated animals undergo a clinicalevaluation every other day to assess cachexia, body weight andlethality.

D-SLAM polypeptides and/or D-SLAM agonists of the invention may also beexamined in this acute GVHD murine model.

It will be clear that the invention may be practiced otherwise than asparticularly described in the foregoing description and examples.Numerous modifications and variations of the present invention arepossible in light of the above teachings and, therefore, are within thescope of the appended claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, laboratory manuals, books, orother disclosures) in the Background of the Invention, DetailedDescription, and Examples is hereby incorporated herein by reference.Moreover, the sequence listing is herein incorporated by reference.

Further, the Sequence Listing submitted herewith, and the SequenceListings submitted in U.S. application Ser. Nos. 10/062,523, filed Feb.5, 2002, Ser. No. 09/369,248, filed Aug. 5, 1999, and Ser. No.09/244,110, filed Feb. 4, 1999, in U.S. Provisional Application Ser. No.60/267,523, filed Feb. 6, 2001, 60/190,062 filed Mar. 17, 2000,60/073,962, filed Feb. 6, 1998, and 60/078,572, filed Mar. 19, 1998, andin International Patent Application Serial No. PCT/US00/21130, filedAug. 3, 2000, in both computer and paper forms in each case, are herebyincorporated by reference in their entireties.

1. An isolated nucleic acid molecule comprising a polynucleotide havinga nucleotide sequence at least 95% identical to a sequence selected fromthe group consisting of: (a) a polynucleotide fragment of SEQ ID NO:1 ora polynucleotide fragment of the cDNA sequence included in ATCC DepositNo: 209623; (b) a polynucleotide encoding a polypeptide fragment of SEQID NO:2 or the cDNA sequence included in ATCC Deposit No: 209623; (c) apolynucleotide encoding a polypeptide domain of SEQ ID NO:2 or the cDNAsequence included in ATCC Deposit No: 209623; (d) a polynucleotideencoding a polypeptide epitope of SEQ ID NO:2 or the cDNA sequenceincluded in ATCC Deposit No: 209623; (e) a polynucleotide encoding apolypeptide of SEQ ID NO:2 or the cDNA sequence included in ATCC DepositNo: 209623 having biological activity; (f) a polynucleotide which is avariant of SEQ ID NO: 1; (g) a polynucleotide which is an allelicvariant of SEQ ID NO:1; (h) a polynucleotide which encodes a specieshomologue of the SEQ ID NO:2; and (i) a polynucleotide capable ofhybridizing under stringent conditions to any one of the polynucleotidesspecified in (a)-(h), wherein said polynucleotide does not hybridizeunder stringent conditions to a nucleic acid molecule having anucleotide sequence of only A residues or of only T residues.
 2. Theisolated nucleic acid molecule of claim 1, wherein the polynucleotidefragment comprises a nucleotide sequence encoding a mature form or asecreted protein.
 3. The isolated nucleic acid molecule of claim 1,wherein the polynucleotide fragment comprises a nucleotide sequenceencoding the sequence identified as SEQ ID NO:2 or the coding sequenceincluded in ATCC Deposit No:
 209623. 4. The isolated nucleic acidmolecule of claim 1, wherein the polynucleotide fragment comprises theentire nucleotide sequence of SEQ ID NO:1 or the cDNA sequence includedin ATCC Deposit No:
 209623. 5. The isolated nucleic acid molecule ofclaim 2, wherein the nucleotide sequence comprises sequential nucleotidedeletions from either the C-terminus or the N-terminus.
 6. The isolatednucleic acid molecule of claim 3, wherein the nucleotide sequencecomprises sequential nucleotide deletions from either the C-terminus orthe N-terminus.
 7. A recombinant vector comprising the isolated nucleicacid molecule of claim
 1. 8. A method of making a recombinant host cellcomprising the isolated nucleic acid molecule of claim
 1. 9. Arecombinant host cell produced by the method of claim
 9. 10. Therecombinant host cell of claim 9 comprising vector sequences.
 11. Anisolated polypeptide comprising an amino acid sequence at least 95%identical to a sequence selected from the group consisting of: (a) apolypeptide fragment of SEQ ID NO:2 or the encoded sequence included inATCC Deposit No: 209623; (b) a polypeptide fragment of SEQ ID NO:2 orthe encoded sequence included in ATCC Deposit No: 209623 havingbiological activity; (c) a polypeptide domain of SEQ ID NO:2 or theencoded sequence included in ATCC Deposit No: 209623; (d) a polypeptideepitope of SEQ ID NO:2 or the encoded sequence included in ATCC DepositNo: 209623; (e) a mature form of a secreted protein; (f) a full lengthsecreted protein; (g) a variant of SEQ ID NO:2; (h) an allelic variantof SEQ ID NO:2; and (i) a species homologue of the SEQ ID NO:2.
 12. Theisolated polypeptide of claim 11, wherein the mature form or the fulllength secreted protein comprises sequential amino acid deletions fromeither the C-terminus or the N-terminus.
 13. An isolated antibody thatbinds specifically to the isolated polypeptide of claim
 11. 14. Arecombinant host cell that expresses the isolated polypeptide of claim11.
 15. A method of making an isolated polypeptide comprising: (a)culturing the recombinant host cell of claim 14 under conditions suchthat said polypeptide is expressed; and (b) recovering said polypeptide.16. The polypeptide produced by claim
 15. 17. A method for preventing,treating, or ameliorating a medical condition which comprisesadministering to a mammalian subject a therapeutically effective amountof the polypeptide of claim
 11. 18. A method of diagnosing apathological condition or a susceptibility to a pathological conditionin a subject related to expression or activity of a secreted proteincomprising: (a) determining the presence or absence of a mutation in thepolynucleotide of claim 1; and (b) diagnosing a pathological conditionor a susceptibility to a pathological condition based on the presence orabsence of said mutation.
 19. A method of diagnosing a pathologicalcondition or a susceptibility to a pathological condition in a subjectrelated to expression or activity of a secreted protein comprising: (a)determining the presence or amount of expression of the polypeptide ofclaim 11 in a biological sample; and (b) diagnosing a pathologicalcondition or a susceptibility to a pathological condition based on thepresence or amount of expression of the polypeptide.
 20. A method foridentifying binding partner to the polypeptide of claim 11 comprising:(a) contacting the polypeptide of claim 11 with a binding partner; and(b) determining whether the binding partner effects an activity of thepolypeptide.
 21. The gene corresponding to the cDNA sequence of SEQ. IDNO:2.
 22. A method of identifying an activity in a biological assay,wherein the method comprises: (a) expressing SEQ ID NO:1 in a cell; (b)isolating the supernatant; (c) detecting an activity in a biologicalassay; and (d) identifying the protein in the supernatant having theactivity.
 23. The product produced by the method of claim 22.